Departament de Producció Vegetal i Ciència forestal
Reconstruction of climatic and crop conditions in the past
based on the isotope signature of archaeobotanical remains
Tesi presentada per Juan Pedro Ferrio Díaz per optar al grau de Doctor
per la Universitat de Lleida.
El present treball ha estat realitzat sota la direcció dels Drs. Jordi Voltas
Velasco (Universitat de Lleida) i Josep Lluis Araus Ortega (Universitat
de Barcelona).
Lleida, Maig 2005
A IRENE
cuando era niña
Agradecimientos
Lejos quedan los tiempos en que la ciencia era cosa de ermitaños que, aislados en parajes remotos, reflexionaban
sobre el mundo y sus dilemas. Hoy en día, no hay progreso científico en el que no intervengan, de una forma u otra,
un buen número de investigadores. Esta tesis (sin asumir que se trate de un progreso científico) no es una excepción,
y es fruto del apoyo y la colaboración de mucha gente. Para empezar, parafraseando a Manolito, “me remontaré al
principio de los tiempos” para agradecer a Luis Tapia la oportunidad de iniciarme en el mundo de la investigación.
Gracias a él entré a formar parte del grupo de José Luis Araus, donde descubrí mi talento en el arte de la
papiroflexia isotópica y, casi simultáneamente, supe de la existencia de unos entes misteriosos llamados “restos
arqueobotánicos”. A José Luis le debo también la posibilidad de dedicar estos años a estudiar “el sexo de los
ángeles”, además de lo mucho que he aprendido sobre la fisiología vegetal, y la confianza que depositó en mí desde
el primer momento. De igual forma, debo agradecer la constancia y el interés mostrado por Jordi Voltas a lo largo de
la tesis. Le doy las gracias también por haberme acogido en Lleida con los brazos abiertos, por su flexibilidad y
comprensión, por ampliar los horizontes de la tesis, por su ayuda con la genética y la estadística, y sobretodo por
compensar con su sensatez mi excesiva tendencia a la divagación.
No cabe decir que un trabajo como este se nutre de la labor de arqueólogos y arqueobotánicos. Las horas de
trabajo que implica la obtención e identificación de los restos se ven apenas reflejadas en las pocas líneas que les he
dedicado en esta tesis. He de agradecer muy especialmente a Natàlia Alonso su implicación constante en el
proyecto, y su paciencia a la hora de responder mis numerosas dudas. Igualmente, me han sido de gran utilidad las
discusiones y las “lecciones prácticas” de arqueobotánica con Ramón Buxó y Georges Willcox. Tampoco olvido la
hospitalidad con que me han recibido siempre Miquel Molist y el resto del equipo de Halula (incluidas “las
macarenas” y el taxista que me acompañó en mi primera prospección). Igualmente hospitalaria fue la recepción del
equipo de Akarçay. Si bien el trabajo realizado allí no se incluye en esta tesis, me ha permitido conocer in situ las
condiciones de trabajo en las excavaciones, lo que me ha ayudado a “mantener los pies en el suelo”.
Por otro lado, la aportación de Pilar Teixidor ha sido fundamental para mi entrada en el mundo de los isótopos.
También he de agradecer a Montse y Mª Josep del departamento de química su ayuda y consejos prácticos.
A Paco, además de agradecerle sus aportaciones técnico-informático-estadísticas, le debo el haber sido
depositario de mis penas y desvaríos, y las veces que entre los dos hemos “arreglado el mundo” (frente a una tesis,
hasta la política es divertida). A Anna, por haber compartido tantas peripecias durante los muestreos de campo, y
otras tantas horas en el laboratorio. A Jaime, por su ayuda en el procesado de las muestras, y por su demostración
empírica de la teoría de la relatividad. Sin olvidar a Alicia y a Luis, por acompañarme en mis “primeros pasos” en el
mundo forestal. Tampoco puedo dejar de mencionar a tantas personas que se cruzaron por mi vida durante mi etapa
en la UB, que me ayudaron a formarme, y han hecho más livianas mis visitas a Barcelona: Edu, Anna, Jordi, Jaume,
Josep, Ricardo, Sabiha, los “Sergis”, las “Evas”, Toni, Salvador, Pere, Lourdes, Santi, Bouchra, Karen... y, con un
recuerdo muy especial, a Hanane. No me olvido de Nieves, Dolors, Jhon, y tantos otros que me ayudaron a
integrarme al llegar a la UdL. Y por supuesto, doy gracias a mis amigos/as del Laboratorio de Entomología, por
“adoptarme” y compartir conmigo el horror cósmico de los lunes a mediodía, además de otros momentos más
agradables: Rosa, Belén, Marcela, Gerardo, Dani, Xavier, Dolors.
Finalmente, he de dar las gracias a mis padres porque, aunque aún no tengan muy claro a qué me dedico (yo
tampoco), sé que siempre les parecerá muy interesante. A mi hermano, por sus ánimos, y por inyectarme un poco de
esa visión práctica de las cosas que yo nunca tendré. A Sergio, por ser “mi amigo de siempre”. A “la Posse”, por sus
frases hechas, y a Juan Manuel, por sus disertaciones sobre cualquier tema. Por supuesto no me olvido de Isabel
(aunque dé más dolores de cabeza que la tesis, en el fondo me cae bien), ni de Inés y la pequeña Lucía, a la que
estoy intentando enseñar la palabra “isótopo”. Pero sin duda, si alguien ha padecido tanto o más que yo estos años,
esa es Irene. Sin su paciencia, su apoyo y sus críticas (a veces hasta constructivas), no hubiera encontrado fuerzas
para llegar hasta aquí. Ni tampoco sin los gruñidos, mordiscos y juegos de mis dos filósofos favoritos...
Good Use Right
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Table of contents
Summary___________________________________________________________________________ 9
Resum ____________________________________________________________________________ 10
Resumen __________________________________________________________________________ 11
General Introduction
I. Overview ________________________________________________________________ 15
II. Stable isotopes in plant science ____________________________________________ 17
II.1 Theory and nomenclature_________________________________________________________ 17
II.2 Carbon isotopes in plant physiology_________________________________________________ 19
II.2.1 Physiological basis of carbon isotope studies in plants _____________________________ 20
II.2.2 Environmental conditions reflected in δ13C ______________________________________ 21
Light intensity__________________________________________________________________ 22
Atmospheric CO2 concentration____________________________________________________ 23
Water availability and water demand ________________________________________________ 23
II.3 Oxygen isotopes in plant physiology_________________________________________________ 24
II.3.1 Environmental conditions reflected in δ18O ______________________________________ 25
III. Palaeenvironmental studies _______________________________________________ 27
III.1. How can we study past climates? __________________________________________________ 27
III.1.1. Historical climatology ______________________________________________________ 27
III.1.2. Ice-cores _________________________________________________________________ 27
III.1.3. Pollen records _____________________________________________________________ 28
III.1.4. Lake and ocean sediments ___________________________________________________ 28
III.1.5. Coral reefs________________________________________________________________ 29
III.1.6. Borehole measurements_____________________________________________________ 29
III.1.7. Tree-rings ________________________________________________________________ 29
From individual tree-ring width series to large-scale chronologies _________________________ 30
Stable isotopes in tree-rings _______________________________________________________ 31
III.2. Holocene climate variability______________________________________________________ 32
1
IV. Archaeobotany _________________________________________________________ 34
IV.1. Main approaches in archaeobotany________________________________________________ 34
IV.2. Some methodological aspects _____________________________________________________ 35
IV.2.1. Preservation of fossil plant remains ___________________________________________ 35
IV.2.2. Recovering of plant remains _________________________________________________ 35
IV.2.3. Dating of samples __________________________________________________________ 36
IV.3. Stable isotopes in archaeobotanical remains_________________________________________ 37
V. Objectives______________________________________________________________ 39
References ________________________________________________________________________ 41
Chapter 1: ∆13C and tree-ring width reflect different drought responses in
Quercus ilex and Pinus halepensis
1.1. Introduction ___________________________________________________________________ 51
1.2. Materials and Methods ___________________________________________________________ 52
1.2.1. Plant material ______________________________________________________________ 52
1.2.2. Tree-ring dating and sample preparation _______________________________________ 53
1.2.3. Meteorological data _________________________________________________________ 54
1.2.4. Carbon isotope discrimination ________________________________________________ 54
1.2.5. Statistical analysis___________________________________________________________ 54
1.3. Results ________________________________________________________________________ 55
1.3.1. ∆13C and growth for Quercus ilex and Pinus halepensis____________________________ 55
1.3.2.Geographic variation ________________________________________________________ 55
1.3.3. Relationship between climatic variables, ∆13C and growth _________________________ 55
Relationship with annual means ___________________________________________________ 55
Relationship with monthly means __________________________________________________ 56
1.4. Discussion _____________________________________________________________________ 57
1.4.1. ∆13C response to water availability and climate __________________________________ 58
1.4.2. Can we track differences in drought response through ∆13C? _______________________ 59
1.4.3. Radial growth response to water availability and climate__________________________ 59
1.4.4. Concluding remarks_________________________________________________________ 60
References ________________________________________________________________________ 61
2
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Chapter 2: Carbon and oxygen isotope ratios in wood constituents of Pinus halepensis
as indicators of precipitation, temperature and vapour pressure deficit
2.1. Introduction____________________________________________________________________ 65
2.2. Materials and Methods ___________________________________________________________ 67
2.2.1. Plant material and sample preparation _________________________________________ 67
2.2.2. Carbon and oxygen isotope analysis ____________________________________________ 67
2.2.3. Meteorological data and δ18O in precipitation ____________________________________ 68
2.2.4. Statistical analysis ___________________________________________________________ 69
2.3. Results and discussion ___________________________________________________________ 70
2.3.1. δ13C and δ18O values in wood and wood fractions ________________________________ 70
2.3.2. Effect of environmental variables on δ13C and δ18O ______________________________ 71
2.3.3. Are δ13C and δ18O complementary? ____________________________________________ 75
2.4. Conclusions ____________________________________________________________________ 76
References ________________________________________________________________________ 77
Chapter 3: Carbon isotope composition of fossil charcoal reveals aridity changes
in the NW Mediterranean Basin
3.1. Introduction____________________________________________________________________ 81
3.2 Materials and methods ____________________________________________________________ 83
3.2.1 Reference plant material: tree-ring dating and sample preparation __________________ 83
3.2.2 Experimental carbonization ___________________________________________________ 83
3.2.3 Archaeological sites and charcoal remains _______________________________________ 84
3.2.4 Carbon concentration and carbon isotope composition_____________________________ 84
3.2.5 Meteorological data __________________________________________________________ 85
3.2.6 Statistical analyses ___________________________________________________________ 85
3.3 Results_________________________________________________________________________ 86
3.3.1 Effect of carbonization on wood δ13C and %C ___________________________________ 86
3.3.2 δ13C and %C in archaeological charcoal _________________________________________ 87
3.3.3 Estimation of past water availability from the ∆13C of archaeological charcoal _________ 88
3.4 Discussion______________________________________________________________________ 90
3.4.1 Is the climatic signal of wood δ13C preserved in charcoal? __________________________ 90
3
3.4.2 %C in charcoal as indicator of carbonization level ________________________________ 90
3.4.3 Changes in aridity during the last 4000 years in the NW Mediterranean Basin_________ 91
3.5 Conclusions ____________________________________________________________________ 93
References ________________________________________________________________________ 94
Chapter 4: Water management practices and climate in ancient agriculture: inference
from the stable isotope composition of archaeobotanical remains
4.1. Introduction ___________________________________________________________________ 99
4.2. Materials and Methods __________________________________________________________ 101
4.2.1. Review on δ13C data from archaeological grains_________________________________ 101
4.2.3. Reference ∆13C values in present crops ________________________________________ 101
4.2.4. Meteorological data ________________________________________________________ 101
4.2.5. Statistical analysis__________________________________________________________ 101
4.3. Results and Discussion __________________________________________________________ 104
4.3.1. Modelling water inputs from ∆13C in grains ____________________________________ 104
4.3.2. Applications of ∆13C to fossil plant remains_____________________________________ 105
Reconstruction of past climate changes in the Mediterranean Basin_______________________ 105
Is it feasible to discriminate between climatic and anthropogenic effects on ∆13C? ___________ 106
4.4. Conclusions___________________________________________________________________ 109
References _______________________________________________________________________ 110
Chapter 5: Estimating grain weight in archaeological cereal crops: a quantitative
approach for comparison with current conditions
5.1. Introduction __________________________________________________________________ 115
5.2. Experimental procedure _________________________________________________________ 116
5.2.1. Plant material and charring conditions ________________________________________ 116
5.2.2. Grain measurements _______________________________________________________ 116
5.2.3. Statistical analyses _________________________________________________________ 117
5.3. Results and discussion __________________________________________________________ 118
5.3.1. Changes in grain size and morphology during carbonisation ______________________ 118
5.3.2. Sample preservation: does initial grain weight affect sample survival during charring? 119
5.3.3. Grain weight models: performance and range of application ______________________ 119
4
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5.4. A case study with archaeological samples ___________________________________________ 121
5.5. Conclusions ___________________________________________________________________ 123
References _______________________________________________________________________ 124
Chapter 6: Evolution of grain weight in archaeological cereal seeds: potential role of
climate and genetic improvement
6.1. Introduction___________________________________________________________________ 129
6.2. Materials and Methods __________________________________________________________ 131
6.2.1. Archaeological sites and plant remains_________________________________________ 131
6.2.2. Carbon isotope composition and meteorological data_____________________________ 131
6.2.3. Estimation of grain weight ___________________________________________________ 132
6.2.4. Estimation of water inputs during grain filling __________________________________ 132
6.2.5. Statistical analyses _________________________________________________________ 132
6.3. Results and Discussion __________________________________________________________ 133
6.3.1 Grain weight and carbon isotope values in archaeological material__________________ 133
6.3.2 Evolution of carbon isotopes and grain weight during the last 4000 years ____________ 135
6.3.3 To what extent water stress conditioned grain weight in ancient crops? ______________ 135
6.3.4 Climatic and genetic effects on the water status of cereal crops _____________________ 136
6.4. Conclusions ___________________________________________________________________ 137
References _______________________________________________________________________ 138
General Discussion
I. Different roads to Rome: evolutionary keys for the divergences between Aleppo pine
and Holm oak in response to drought __________________________________________________ 143
II. Sensitivity to seasonal climate of stable isotopes in tree-rings: implications for
(palaeo)environmental studies________________________________________________________ 146
Reinterpreting the “Cold Iron Age Epoch” in the Ebro Basin on the light of ∆13C data______ 148
III. Time course of CO2 during the Holocene and the use of carbon isotopes in plant
material as palaeoenvironmental tools _________________________________________________ 149
IV. Reconstruction of plant growing conditions in ancient crops: main limitations
and future prospects ________________________________________________________________ 150
To what extent are grain weight estimations a reliable source to study changes in
potential (i.e. genetic) grain size of ancient crops ? ____________________________________ 150
5
Genotype effects on water availability and ∆13C ______________________________________ 151
Traditional practices and water availability of crops __________________________________ 151
Considering other environmental factors: nutrient availability and temperature __________ 152
V. Concluding remarks _____________________________________________________________ 154
References _______________________________________________________________________ 155
Conclusions
I. Reconstruction of climate _________________________________________________________ 163
II. Reconstruction of crop conditions __________________________________________________ 165
Table index_______________________________________________________________________ 169
Figure index______________________________________________________________________ 171
6

Summaries
7
Summary
Reconstruction of climatic and crop conditions in the past
based on the isotope signature of archaeobotanical remains
The onset and progressive development of agriculture during the last 10,000 years has modified radically the
social and demographic structure of human groups, as well as their interaction with the environment. In fact, the
adoption of agriculture is probably the first example of reciprocal interaction between the environment and the
humanity. Thus, reconstructing the environmental conditions (either climatic or anthropogenic) that characterised
this process is of great interest in order to know their potential causes, as well as to understand the long-term effects
of agriculture economy on the environment. The general objective of this Thesis is the development of new tools
addressed to the reconstruction of climatic and crop conditions during the origins and spread of agriculture in
Prehistory. The proposed methodology is based on the study of plant remains (wood charcoal and charred seeds)
routinely recovered during archaeological excavations, with special emphasis on the use of stable isotopes as
palaeoenvironmental indicators. The work can be divided into two main blocks. The first attempts to get a picture of
the climatic context in which evolved agriculture, while the second one is centred on the specific growing conditions
of cultivated crops.
The objective of the first bock is to obtain a methodology allowing the quantification of aridity changes in the
past from the stable isotope analysis of wood charcoal from forest species. This requires a previous calibration with
modern material, as well as the assessment of the effect of carbonisation on the stable isotope signature of wood.
The first chapter characterises the climatic signature in carbon isotope composition (δ13C) of wood in Mediterranean
conditions, using Pinus halepensis Mill. and Quercus ilex L. as reference species, finding that in both species δ13C is
mostly related to precipitation. The second chapter studies the relationship between climate and oxygen isotope
composition (δ18O) for P. Halepensis, as well as the differences in the isotopic signature (δ13C and δ18O) in the two
major components of wood: cellulose and lignin. We found that the climatic signal of δ13C is equally present in
cellulose and lignin, whereas for δ18O cellulose extraction is necessary to obtain a climatic signal, which prevents its
application in carbonised material. Finally, the third chapter shows how carbonisation, although affecting
significantly the δ13C of wood, does not remove the original climatic signal, which can be recovered by means of a
simple correction, taking carbon concentration as a reference variable. Using this methodology, we reconstructed
aridity changes in the Cinca and Segre valleys (Ebro Depression) during the last 4,000 years, finding greater water
availability in the last millennia than in present.
The second block describes different aspects of crop conditions in prehistoric agriculture, combining δ13C
analyses with morphometrical variables of cultivated species, mostly cereals. Chapter four constitutes a critical
review of the isotopic data available for archaeobotanical remains, proposing new approaches in order to maximise
the information retrieved from these data. It shows how the combined analysis of crop and forest species, as well as
the comparison among different crops, may help to detect human-driven changes in crop water availability. Chapter
five develops a new methodology to estimate grain weight in cereals cultivated in the past, from charred grain
dimensions, and considering the effect of carbonisation. This allows, for the first time, a direct comparison between
archaeobotanical data and current agronomic studies. Finally, Chapter six combines data obtained using the methods
described in the three former chapters to study the potential causes of the observed grain weight changes in Cinca
and Segre valleys during the last 4,000 years.
9
Resum
Reconstrucció de les condicions climàtiques i de conreu en el passat
a partir de l’anàlisi isotòpica de restes arqueobotàniques
L’aparició i progressiu desenvolupament de l’agricultura durant els darrers 10.000 anys ha alterat radicalment
l’estructura social i poblacional dels grups humans, així com la seva relació amb l’entorn. De fet, l’adopció de
l’agricultura pot constituir el primer exemple en el temps d’interacció recíproca entre el medi ambient i la humanitat.
Per tant, la reconstrucció de les condicions ambientals (d’origen climàtic o degudes a l’acció humana) que van
caracteritzar aquest procés és de gran interès per tractar de conèixer-ne les causes, així com per entendre els efectes
a llarg termini que ha tingut l’economia agrícola en el medi. L’objectiu general de la tesi és desenvolupar noves
eines destinades a reconstrucció de les condicions climàtiques i de conreu durant l’origen i dispersió de l’agricultura
a la prehistòria. La metodologia que es proposa està basada en l’estudi de les restes vegetals (fustes i llavors
carbonitzades) que són recuperades rutinàriament en excavacions arqueològiques, posant especial èmfasi en l’ús
dels isòtops estables com a indicadors paleoambientals. El treball es pot dividir en dos grans blocs. El primer pretén
configurar l’entorn climàtic en el que va evolucionar l’agricultura, en tant que el segon se centra en la caracterització
de les condicions específiques de creixement de les espècies conreades.
El primer bloc té com a objectiu la posada a punt d’una metodologia que permeti quantificar els canvis d’aridesa
en el passat, a partir de l’anàlisi isotòpica de carbons d’espècies forestals. Això requereix una calibració prèvia amb
material actual, així com una valoració de l’efecte de la carbonització sobre la signatura isotòpica de la fusta. Al
primer capítol, es va caracteritzar la signatura climàtica present a la composició isotòpica de carboni (δ13C) de la
fusta en condicions mediterrànies, fent servir Pinus halepensis Mill. i Quercus ilex L. Com a espècies de referència,
trobant que en totes dues espècies aquesta es podia relacionar sobretot amb la precipitació. Al segon capítol, es va
estudiar la relació entre el clima i la composició isotòpica d’oxigen (δ18O) per P. halepensis, així com les diferències
en la signatura isotòpica (δ13C i δ18O) present als dos components fonamentals de la fusta: cel⋅lulosa i lignina. Es va
trobar que la component climàtica de la δ13C és igualment present a cel⋅lulosa i lignina, en tant que per la δ18O
l’extracció de cel⋅lulosa és necessària per tal d’obtenir informació climàtica, fet que impedeix la seva aplicació en
material arqueològic. Finalment, al tercer capítol es va poder demostrar com la carbonització, tot i alterar
significativament la δ13C de la fusta, no n’elimina la component climàtica original, que pot recuperar-se mitjançant
una senzilla correcció, que fa servir el contingut de carboni com a variable de referència. Amb aquesta metodologia
es reconstrueixen els canvis d’aridesa que han tingut lloc a les valls del Cinca i del Segre (Depressió de l’Ebre)
durant els darrers 10.000 anys, trobant una major disponibilitat hídrica al llarg dels darrers mil⋅lenis que no pas en el
present.
El segon bloc aborda la descripció de diferents vessants de les condicions de conreu a l’agricultura prehistòrica,
combinant l’anàlisi de la δ13C amb mesures morfomètriques en llavors d’espècies conreades, fonamentalment
cereals. El capítol quart constitueix una revisió crítica de les dades existent sobre isòtops estables en restes
arqueobotàniques, proposant noves aproximacions per tal de maximitzar la informació que se’n pot derivar. Es
mostra com la combinació d’anàlisis en espècies conreades i forestals, així com la comparació entre diferents
conreus, pot ajudar a detectar canvis en la disponibilitat hídrica dels conreus, fruit de l’acció humana.El cinquè
capítol desenvolupa una nova metodologia que permet estimar el pes del gra dels cereals conreats en el passat, a
partir de les seves dimensions, tot considerant l’efecte de la carbonització. Això permet, per primer cop, establir una
lligam directe entre les dades obtingudes a partir de restes arqueobotàniques i els estudis agronòmics. Finalment, el
capítol sisè combina dades obtingudes a partir dels mètodes descrits en els tres capítols anteriors per resoldre les
possibles causes dels canvis observats en la mida del gra als jaciments les valls del Cinca i del Segre durant els
darrers 4.000 anys.
10

Resumen
Reconstrucción de las condiciones climáticas y de cultivo en el pasado
a partir del análisis isotópico de restos arqueobotánicos
La aparición y progresivo desarrollo de la agricultura durante los últimos 10.000 años ha alterado radicalmente la
estructura social y poblacional de los grupos humanos, así como su relación con el entorno. De hecho, la adopción
de la agricultura puede que constituya el primer ejemplo en el tiempo de interacción recíproca entre el medio
ambiente y la humanidad. De ahí que reconstruir las condiciones ambientales (de origen climático o debidas a la
acción humana) que caracterizaron este proceso sea de gran interés para tratar de conocer sus posibles causas, así
como para entender los efectos a largo plazo que ha tenido la economía agrícola en el medio. El objetivo general de
esta tesis es el desarrollo de nuevas herramientas destinadas a reconstruir las condiciones climáticas y de cultivo
durante el origen y dispersión de la agricultura en la prehistoria. La metodología propuesta se basa en el estudio de
los restos vegetales (maderas y semillas carbonizadas) que son recuperados rutinariamente en excavaciones
arqueológicas, poniendo especial énfasis en el empleo de los isótopos estables como indicadores paleoambientales.
El trabajo se puede dividir en dos grandes bloques. El primero pretende configurar el entorno climático en el que
evolucionó la agricultura, mientras que el segundo se centra en caracterizar las condiciones específicas de
crecimiento de las especies cultivadas.
El primer bloque tiene como objeto la puesta a punto de una metodología que permita cuantificar los cambios de
aridez en el pasado, partiendo del análisis isotópico de carbones de especies forestales. Esto requiere una calibración
previa con material actual, así como una valoración del efecto de la carbonización en la señal isotópica de la madera.
En el primer capítulo, se caracterizó la señal climática presente en la composición isotópica de carbono (δ13C) de la
madera en condiciones mediterráneas, empleando Pinus halepensis Mill. y Quercus ilex L. como especies de
referencia, encontrando que en ambas especies ésta se podía relacionar fundamentalmente con la precipitación. En el
segundo capítulo, se estudió la relación entre el clima y la composición isotópica de oxígeno (δ18O) para P.
halepensis, así como las diferencias en la señal isotópica (δ13C y δ18O) presente en los dos componentes principales
de la madera: celulosa y lignina. Se encontró que la señal climática de la δ13C es igualmente presente en celulosa y
lignina, mientras que para la δ18O la extracción de celulosa es necesaria para obtener una información climática, lo
que impide su aplicación en material carbonizado. Finalmente, en un tercer capítulo se demostró cómo la
carbonización, si bien afecta significativamente la δ13C de la madera, no elimina la señal climática original, que
puede recuperarse mediante una simple corrección, incluyendo el contenido de carbono como variable de referencia.
Mediante este metodología se reconstruyen los cambios de aridez ocurridos en los valles del Cinca y del Segre
(Depresión del Ebro) durante los últimos 4.000 años, observando una mayor disponibilidad hídrica en los últimos
milenios en relación con el presente.
El segundo bloque se centra en describir diferentes aspectos de las condiciones de cultivo en la agricultura
prehistórica, combinando el análisis de la δ13C con medidas morfométricas en semillas de especies cultivadas,
fundamentalmente cereales. El capítulo cuarto constituye una revisión crítica de la información isotópica existente
sobre restos arqueobotánicos, proponiendo nuevos enfoques destinados a maximizar la información extraíble a partir
de estos datos. Se muestra cómo la combinación de análisis en especies cultivadas y forestales, así como la
comparación entre cultivos, puede ayudar a detectar cambios en la disponibilidad hídrica de los cultivos debidos a la
acción humana. El capítulo quinto desarrolla una nueva metodología que permite estimar el peso del grano en los
cereales cultivados en el pasado, a partir de sus dimensiones, y considerando el efecto de la carbonización. Esto
permite, por primera vez, una comparación directa entre los datos obtenidos a partir de restos arqueobotánicos y la
bibliografía agronómica. Finalmente, el capítulo sexto combina datos obtenidos mediante los métodos descritos en
los tres capítulos anteriores para resolver las posibles causas de los cambios en el tamaño del grano observados en
los valles del Cinca y del Segre durante los últimos 4.000 años.
11
General Introduction
“Kung Tsé preguntó a Yeu:
¿Sabes en qué consiste la verdadera ciencia? En conocer que se sabe
lo que realmente se sabe, y que se ignora lo que en verdad se ignora.”
Kung Tsé. Lun Yu
13
Introduction
I. Overview
During the Holocene, agriculture has been adopted in many parts of the world as independent
processes: till now, several archaeological evidences have been found for plant domestication in the Near
East, Ethiopia, sub-Saharan Africa, China, Mesoamerica and South America. In particular, western
agriculture, based on cereals (wheat and barley) and legumes (pea and lentil), started in an area of the
Near East known as the “Fertile Crescent”, comprising the planes of Mesopotamia, the deserts of Syria
and Palestine, moreover to some mountain areas of Anatolia. Around the world, agriculture appeared
during the last 10,000 years, a few millennia after the retreat of Pleistocene ice, spreading during what has
been the most stable warm period in almost half a million years. Thus, the origins and subsequent
expansion of agriculture cannot be isolated from the particular environmental conditions in which
evolved. A similar reasoning lead V. Gordon Childe to develop, within the concept of “Neolithic
revolution”, the theory that climate changes altered the environment in such a way that forced huntersgatherers to modify their life-style (Childe 1954). Other authors, while embracing the major role of
climate, emphasised the importance of economic changes and cultural and social evolution (Cauvin 1994,
further references in Buxó 1997 and Harlan 1975). It has been also argued that demographic pressure
might have forced this change (Binford 1981; Cohen 1981), but archaeological evidences suggest that
increased population was a consequence rather than a cause of the adoption of agriculture (Harlan 1975;
Harris and Hillman 1989).
As quoted by Harlan (1975) in his “no-model” model for the onset of agriculture, every attempt to
provide a general answer for agricultural origins has generated evidence against it, and the most probable
explanation is that plant cultivation began in different areas for different reasons. What seems undoubtful
is that climate have had always a major role in the success of early farming communities, given that, even
in today’s most advanced agricultural exploitations, climate is the main factor determining crop yields.
On the other hand, the spread of agriculture caused changes in landscape and environment without
precedent in the history of human-environment interaction. Agriculture can be associated not only to
current deforestation and erosion in many areas in the world, but also might have altered significantly the
course of climate, initially at the local scale, but ultimately at a global dimension. Thus, the adoption and
spread of agriculture might be considered the first example of reciprocal interaction between climate and
the humanity. In such context, reconstructing the environmental conditions prevailing in prehistoric
agriculture is of great relevance. On the one hand, it may help to understand the context in which
agriculture developed, and on the other, it may be useful to attain information regarding the long-term
environmental effects of agriculture in the past.
Past global climate changes during the Holocene have been well defined through a variety of climate
proxies (Committee on abrupt climate change 2002; Folland et al. 2001). However, there is a need to
concentrate efforts at the local scale, in order to attain a clearer picture of the specific climatic conditions
faced by early farmers. This is particularly true for precipitation, as it shows a considerable spatial
heterogeneity (Magny et al. 2003). The general objective of this Thesis is to develop new tools addressed
to 1) reconstruct the specific climatic conditions (mostly in terms of water availability) in which ancient
agriculture was developed, providing information at a local scale, and 2) to determine the use of different
15
Introduction
agronomic practices addressed to improve yields and crop water availability. The methodology proposed
is based on the study of carbonised plant remains (wood charcoal and charred seeds) that are routinely
recovered in the course of archaeological excavations. Although emphasis is mainly put on the use of
stable isotopes as palaeoenviromental indicators, the Thesis takes also advantage of the information
provided by several biomorphometric parameters. The work can be structured in two main blocks:
BLOCK 1. Reconstruction of climate
The general objective is to develop a new methodology in order to quantify past aridity changes
through the analysis of stable isotopes in wood charcoal. This block includes 3 chapters:
• Chapter 1 compares the effect to climate and site variables on carbon isotope composition and
radial growth for two Mediterranean trees, Pinus halepensis Mill. and Quercus ilex L.
• In Chapter 2 the climatic relationships of P. halepensis are further studied, considering both
carbon and oxygen isotopes, in wood and its two main components, lignin and cellulose.
• Finally, Chapter 3 assess the effect of carbonisation on the carbon isotope composition of wood
from P. halepensis, and presents a case study to reconstruct aridity changes during the last four
millennia in the Ebro Basin from the analysis of fossil charcoal.
BLOCK 2. Reconstruction of crop conditions
This block aims to describe different aspects of crop conditions in prehistoric agriculture, combining
the analysis of carbon isotopes with morphometric measurements in archaebotanical remains of cultivated
species, mostly cereals. It is also divided into three chapters:
• Chapter 4 is a critical review of current isotopic data regarding archaeobotanical remains,
proposing new ways to deal with these data, through the combination of analysis in cultivated and
forest species, as well as by comparing different crops.
• In Chapter 5 a new methodology is developed in order to obtain a quantitative estimation of
cereal grain weight in the past from grain dimensions, bearing in mind the effect of carbonisation.
• Finally, Chapter 6 combines data obtained from the methodologies described in the three former
chapters to solve the possible causes of the observed changes in grain weight during the last four
millennia in the Ebro Basin.
16

Introduction
II. Stable isotopes in plant science
II.1 Theory and nomenclature
Isotopes are atoms of the same element that have different numbers of neutrons. Differences in the
number of neutrons among the various isotopes of an element mean that the various isotopes have
different masses. The superscript number to the left of an element designation is called the mass number
and is the sum of the number of protons and neutrons in the isotope. For example, all isotopes of oxygen
have 8 protons; however, an oxygen atom with a mass of 18 (denoted 18O) has 2 more neutrons than an
oxygen atom with a mass of 16 (16O). Isotopes can be divided into radioactive and non-radioactive. The
former disintegrate spontaneously over time to form other isotopes, whereas the latter do not appear to
decay to other isotopes on geologic time scales, and thus are also known as stable isotopes.
The most abundant elements in the biosphere are carbon (C), hydrogen (H), oxygen (O) and nitrogen
(N), being 13C, 2H (D), 18O and 15N the stable isotopes of greater interest in plant physiology. The stable
isotope composition of a given sample is determined by mass spectrometry, an is usually expressed in
diffential notation:
Eq. I
δ (‰) = ( Rsample / Rstandard − 1) × 1000
where δ stands for the isotopic composition, in parts per mil (‰),as referred to an standard (see Table
I). R denotes the ratio of the heavy to light isotope (e.g., 13C/12C), and Rsample and Rstandard are the ratios in
the sample and standard, respectively. A positive δ value means that the isotopic ratio of the sample is
higher than that of the standard; a negative δ value means that the isotopic ratio of the sample is lower
than that of the standard. For example, a δ13C value of -28‰ means that the 13C/12C of the sample is 28
parts-per-thousand lower than the 13C/12C of the standard (Pee-Dee Belemnite limestone).
Isotopic composition of a given element varies considerably between the different pools of the
biosphere. This phenomenon is called isotopic fractionation, and is determined by isotope effects
occurring during the cleavage or formation of atomic bonds, as well as during other processes affected by
atomic mass (e.g. diffusion). Thus, some substances are enriched in the heavier isotope, while others
become depleted (i.e. lighter). We can distinguish two kinds of isotopic effects: kinetic ad
thermodynamic. The former are due to differences between isotopes in the rate of a given reaction and the
latter reflect divergences in the equilibrium constants of the reaction. Kinetic isotope effects of a chain of
17
Introduction
reactions are generally non-additive, whereas thermodynamic effects are additive. Since isotope effects
have values usually very close to unity, they are often expressed in terms of isotopic discrimination (∆),
defined as its deviation from unity:
Eq. II
∆ (‰ ) = α − 1 =
δr − δp
1 + δp / 1000
where α is the isotope effect associated with the reaction, and δr and δp stand for the isotopic
composition of reactives and products, respectively. Moreover of being a more intuitive expression of the
consequences of a given process, it allows an easier comparison of the results obtained by different
researchers.
Table I Standards, notation, abundance, typical range in plants and mean analytical error of the stable isotopes most commonly
used in plant physiology. Adapted from Mateo et al. (2004). Original data from Barbour et al. (2001), Ehleringer and Rundel
(1988), Epstein et al. (1977), Farquhar et al. (1989) and Handley and Raven (1992).
Isotope pair
13
Standard
Differential notation
Mean abundance (%)a
Observed range δ (‰)
Analytical errord (‰)
a
C/12C
PDBb
δ13C
1,1
-35 to -5
0.1
15
N/14N
Air N2
δ15N
0,37
-10 to +10
0.2
18
O/16O
SMOWc
δ18O
0,20
+15 to +35
0.05-0.2
Abundances of the heavier isotope against the total pool of the element
PDB, Pee-Dee Belemnite (limestone): already used up, replaced by secondary standards
c
SMOW, Standard Mean Ocean Water
d
Overall analytical precision (standard error: sample preparation + internal error of mass spectrometer)
b
18

2
H(D)/1H
SMOWc
δD
0,015
-300 to +20
4-7
Introduction
II.2 Carbon isotopes in plant physiology
The isotopic family of carbon includes two stable isotopes (12C and 13C) and several radioactive
isotopes. Carbon is the most abundant element in the biosphere and, moreover, participates in virtually
any of the biological reactions. Indeed, the study of carbon isotope composition (δ13C) provides useful
information about several physiological processes and is particularly useful as an integrative
measurement of plant response against the environment. On the other hand, 13C is relatively abundant
(Table I) which, along with its wide range of variation, makes its determination easier, allowing very
precise results at a relatively low cost.
The range of variation of δ13C in nature is greater than 100‰ (Ehleringer and Rundel 1988). However,
the most usual values in the geosphere and biosphere remains between –40‰ and 0‰ (see Fig. I).
Average δ13C of atmospheric CO2 is -8‰, although this value is becoming more negative year by year
(ca. 0.02-0.03‰/year) due to the synergic effect of deforestation and the use of fossil fuels (Keeling et al.
1979). Moreover, there is an important seasonal variation of atmospheric δ13C, which is determined by
the vegetation cover, being far clearer in the Northern than in the Southern hemisphere (Fig. II). Such
divergence is caused by the fact that, in Northern hemisphere, vegetation is concentrated in medium to
high latitudes, and thus exposed to seasonal cycles of activity, whereas Southern hemisphere vegetation is
dominated by the equatorial rainforest. Atmospheric δ13C variations (seasonal or interanual) should be
considered when comparing species differing in their growing cycles, as well as distant samples, either in
Fig. I Range of carbon isotopic composition (δ13C) within the main compartments of environment and biosphere. Redrawn from
Mateo et al. (2004). Original data from Ehleringer and Rundell (1988) and Vogel (1993).
19
Introduction
Mace Head, Ireland (53ºN)
Ragged Point, Barbados (13ºN)
Tierra del Fuego, Argentina (55ºS)
-7.2
δ13C air
-7.4
-7.6
-7.8
-8.0
-8.2
-8.4
1995
1996
1997
1998
1999
2000
2001
Year
Fig. II Seasonal and interanual evolution of isotopic composition (δ13C) of atmospheric CO2 in three locations, representative of
Northern Hemisphere, Equatorial Belt and Southern Hemisphere (Mace Head, Ragged Point and Tierra del Fuego, respectively).
For reference, the trend line calculated from all the points and its prediction confidence intervals are plotted. (data from CUINSTAAR and NOAA-CMDL, ftp://ftp.cmdl.noaa.gov/ccg/co2c13/flask/readme.html). Modified from Mateo et al. (2004).
time (at least over 4-5 years) or space (specially latitude). The vicinity (at the regional or local scale) of
important sources of fossil fuel contamination should be also considered. For example, whereas in 1996
the average δ13C in the Malta island was –8.0‰, the same value for Hungary was –8.3‰ (according to
CU-INSTAAR and NOAA-CMDL, ftp://ftp.cmdl.noaa.gov/ccg/co2c13/flask/readme.html).
II.2.1 Physiological basis of carbon isotope studies in plants
The two stable carbon isotopes (13C and 12C) occur in the molar ratio of 1:99 in the atmosphere.
However, plants with C3 photosynthetic pathway generally contain proportionally less 13C than does the
air (Farquhar et al. 1989). Indeed, present δ13C in air is about -8‰, whereas typical C3 leaf composition
becomes -29‰. In many plant physiological studies, carbon isotope discrimination (∆13C) is calculated as
follows (Farquhar et al. 1989):
Eq. III
δ Cair − δ Cplant
δ 13Cplant
(1 +
)
1000
13
∆13C plant =
20

13
Introduction
where δ13Cair and δ13Cplant refer to air and plant composition, respectively. This parameter reflects the
amount in which the heavier isotope 13C is discriminated respect the lighter 12C during the physical and
chemical processes involved in the synthesis of plant organic matter (Farquhar et al. 1989). The main
factors determining ∆13C in C3 plants are diffusion in the air (including the boundary layer and the
stomata) and carbon fixation by the carboxylating enzyme ribulose bisphosphate carboxylase (RuBisCO).
Among the various models developed to describe isotopic discrimination in photosynthesis, the most
extensively used is that of Farquhar et al. (1982):
∆13C ( ‰ ) = a
Ca − Ci
C
C
+ b i = a + (b − a ) i
Ca
Ca
Ca
Eq. IV
where Ca and Ci stand for ambient and intercellular partial pressures of CO2, respectively, a is the
discrimination due to diffusion in air and b is the discrimination due to carboxylation. Therefore, ∆13C
values in C3 plants depend on the relative contribution of these two steps, determined by the relationship
between stomatal conductance and photosynthesis. When stomata are open (fig. IIIa), CO2 diffuses easily
into the intercellular space, and Ci is closer to Ca; thus, ∆13C approaches the value of b (about 30‰). In
other words, RuBisCO is not limited by CO2 and thus discrimination takes place mostly during the
carboxylation step. In contrast, when stomatal conductance is reduced (fig. IIIb), CO2 flux is limited and
Ci is significantly lower than Ca. Therefore, photosynthesis is strongly limited by stomatal conductance,
and ∆13C becomes closer to a, the value of the discrimination during CO2 diffusion in air, (about 4.4‰).
II.2.2 Environmental conditions reflected in δ13C
Following the aforementioned considerations, there are several environmental factors that can modify the
isotopic composition of plant tissues through their influence on either leaf conductance or photosynthetic
rate, or both parameters simultaneously. Changes in irradiance levels, CO2 concentration, and plant water
status (often derived from human activities) are clearly reflected in δ13C variations. However, first
systematic studies about plant δ13C variability were focused on the identification of the recently
discovered C4 and CAM photosynthetic pathways (Ehleringer and Vogel 1993; Troughton 1979). Indeed,
due to the strong correlation between photosynthetic rate and stomatal conductance, it was first
considered that Ci of plants would be constant, depending only on enzymatic and/or anatomical
differences between different kinds of metabolism (Troughton 1979). Later, it was recognised that there is
considerable variation in Ci due to environmental factors, thus reflected in δ13C of plants. Since then,
much effort has been taken to understand the relationship between isotopic composition and
environmental variation.
21
Introduction
Light intensity
Light intensity has been suggested to have a positive relationship with δ13C as, in many studies, δ13C
of leaf tissue mimics irradiance gradients. This is the case, for example, when the vertical profile of δ13C
within a forest canopy is measured (Berry et al. 1997; Francey et al. 1985; Vogel 1978). However,
interpretation of these results has been controversial as it is often difficult to discriminate direct light
effects from those caused by differences in either CO2 concentration, vapour pressure or air δ13C
throughout the canopy. Vogel (1978) attributed this “canopy effect” to a recycling of soil CO2, which had
lower (about 12‰) δ13C than atmospheric CO2. However, in those studies where simultaneous
Fig. III Simplified scheme of the relationship between carbon isotope discrimination (∆13C) in C3 plants and stomatal
conductance. A) High stomatal conductance, high discrimination: CO2 diffusses easily into the intercelular space, the activity of
the carboxylating enzyme (RuBisCO) is not limited by CO2 concentration and thus it has more chances to discriminate against
13
C. B) Low stomatal conductance, low discrimination: the flux of CO2 is reduced, and the limiting factor of photosynthesis is
stomatal conductance. In this case RuBisCO is forced to fix a higher proportion of 13C. Redrawn from Ferrio et al. (2003).
22

Introduction
measurement of air δ13C were performed (Berry et al. 1997; Francey et al. 1985), plant δ13C was
significantly correlated with sampling height, without a corresponding change in air δ13C. Therefore, most
of the decrease in leaf δ13C with canopy depth is likely to be related with stomatal and photosynthetic
effects. This does not fully discard CO2 and vapour pressure gradients as potential causes for the observed
δ13C variability within canopies (Broadmeadow et al. 1993).
Other works have shown strong evidence of a direct effect of light on δ13C, at least when this factor is
limiting (Broadmeadow et al. 1993; Yakir and Israeli 1995; Zimmerman and Ehleringer 1990). Taking
advantage of the negative relationship between leaf oxygen isotope composition (δ18O) and transpiration
rates, (Yakir and Israeli 1995) demonstrated that the cause underlying the relationship between δ13C of
banana leaves and irradiance was an increase in photosynthetic capacity, without appreciable variations in
stomatal conductance. Other authors arrived to similar conclusions combining gas-exchange
measurements with δ13C analyses (Broadmeadow et al. 1993). In contrast, Zimmerman and Ehleringer
(1990) sustained that the origin of δ13C increase in orchid leaves at high irradiance was due to declining
stomatal conductance. This conclusion should be taken carefully, as it was only based on the small
differences found in leaf nitrogen content, assuming that this would imply little change in potential
photosynthetic capacity (Lambers et al. 1998).
Atmospheric CO2 concentration
Early in the 60s, Park and Epstein (1960) grew tomato plants at two levels of CO2 and showed that
plants grown at the higher level had a more negative δ13C than plants grown at the lower concentration.
Since then, similar results have been reported for several C3 herbs (Beerling and Woodward 1995; Polley
et al. 1993) and trees (Beerling 1997; Picon et al. 1997) over a wide range of CO2 concentrations. In C3
plants, CO2 is usually limiting photosynthesis and, thus, an increase in CO2 results in greater
photosynthetic rates. On the other hand, plants take advantage of the increased CO2 availability to
augment water use efficiency (i.e. the ratio between net assimilation and water transpired) by closing
stomata. Experimental results indicate that this reduction in stomatal conductance does not limit
photosynthesis, thus δ13C values become more negative as CO2 concentration increases (Beerling and
Woodward 1995; Polley et al. 1993). Therefore CO2 and the above-mentioned light gradients may have
additive effects within closed canopies, both contributing to leaf δ13C decrease with depth (Berry et al.
1997; Broadmeadow et al. 1993).
Water availability and water demand
Plants typically react against a decrease in water availability through stomata closure and, although
carboxylation rates may also decline under water shortage, leaf conductance is usually affected to a larger
extent, originating a reduction in Ci and a concomitant increase in δ13C (Farquhar et al., 1989)
(Scheidegger et al. 2000). Many studies under growth-chamber and field conditions have shown that
plants developed under water stress (stress induced by low soil water content) produced leaves with
higher δ13C (see references in Ferrio et al. 2003; Griffiths and Parry 2002; Mateo et al. 2004; Warren et
23
Introduction
al. 2001). On the other hand, it is leaf water availability the factor that ultimately influences δ13C and this
availability depends not only on the water input from the soil, but also on its physical structure as well as
on the hydraulic resistance along the plant xylem (Masle and Farquhar 1988; Warren and Adams 2000).
Moreover, the rate of evaporation from the leaf also determines stomatal responses that subsequently
affect δ13C. Indeed, an increase in the leaf-to-air vapour pressure gradient (VPG, which is considered the
driving force for transpiration) will also cause a reduction in Ci, leading to higher δ13C values (Barbour
and Farquhar 2000; Ehleringer 1990). In accordance with these assumptions, it should be expected to find
significant relationships between δ13C and environmental parameters related with water availability or
VPG, such as precipitation, relative humidity or potential evapotranspiration. Stewart et al. (1995), for
example, analysed δ13C from 12 plant communities along a rainfall gradient, finding significant negative
correlations between annual rainfall and community-averaged δ13C. Similar relationships with water
availability have been reported over smaller scales, reflecting microenvironmental changes (Ehleringer
and Cooper 1988; Peñuelas et al. 1999). On the other hand, the expected positive correlation between
VPG and δ13C along climate gradients has also been reported elsewhere (Sparks and Ehleringer 1997;
Williams and Ehleringer 1996).
It is important to note here, however, that in most cases the relationship between δ13C and plant water
availability is not linear, showing a saturation trend as water availability increases (Araus et al. 1997a;
Korol et al. 1999; Warren et al. 2001). Warren et al. (2001), for example, found that, over a global survey
of δ13C values on conifers, δ13C reached an asymptotic value once there is no water deficit, namely when
the ratio between precipitation and evapotranspirative demand equalled unity. The reason for that general
trend is that the main factor relating δ13C with water inputs is stomatal conductance, which is expected to
show progressively smaller changes as plants become less stressed. Under such conditions, other factors,
such as altitude (Hultine and Marshall 2000), irradiance (Leavitt and Long 1991; Warren et al. 2001) or
even nitrogen availability (Lopes et al. 2004; Peñuelas et al. 1999) might have as large an effect on δ13C
as climatic variables affecting plant water status, such as precipitation or evapotranspirative demand.
II.3 Oxygen isotopes in plant physiology
The interpretation of oxygen isotope composition (δ18O) variability in plants is not as direct as that of
δ13C, as it involves a combination of abiotic and biotic fractionation processes. The study of oxygen
isotopes has been traditionally linked to that of the other element of water, hydrogen. In 1932, Urey et al.
(1932) discovered a heavier form of hydrogen: Deuterium (2H). Using measures of water density, they
found considerable variations among samples of different origins. Years after the discovery of Urey,
Dansgaard (1964) began a systematic analysis of 18O and 2H in marine and continental waters, finding
that the first were more enriched in the heavier isotopes than the latter. On the other hand, the relationship
between δ18O and δ2H in the world’s fresh waters follows a predictable linear relationship, referred to as
the “meteoric water line” (Craig 1961).
24

Introduction
II.3.1 Environmental conditions reflected in δ18O
Craig’s meteoric water line is a direct consequence of the fact that fresh waters of warm regions have
more enriched (positive) values of hydrogen and oxygen isotopes, whereas cool regions are isotopically
depleted for both elements. Such differences are mostly due to fractionation occurring during phase
changes in the course of the hydrological cycle (see Fig. IV). On the one hand, light isotopes (16O and 1H)
evaporate more rapidly than their heavier counterparts, and thus water vapour is isotopically depleted
respect source water (e.g. ocean water). On the other, the opposite occurs during precipitation. In order
for water to condense and precipitate from an air mass, the temperature must drop. As temperature
decreases, the heavier isotopes are selectively precipitated through distillation. This phenomenon is
referred to as the “rainout” effect (Dansgaard 1964), which describes the successive depletion as air mass
travels from its source and loses heavier isotopes. This effect can be enhanced by orographic effects that
may cause an abrupt cooling of an air mass as it rises. On the other hand, the amount of this fractionation
varies according to temperature and, as a consequence, isotopic composition of precipitation is positively
correlated with temperature.
The source of water for most terrestial plants is soil moisture, so part of the signal in water isotopes (H
and O) of plants come from the isotopic signature of precipitation. However, there are several potential
fractionations before the water isotopes become fixed in plant tissues (Fig. IV). The first occurs within the
soil, as evaporation affects the original isotopic signal, so the residence time and deep of soil water is
important (Buhay and Edwards 1995). On the other hand, the observed discrepancy between the δ18O of
molecular oxygen in atmosphere (of photosynthetic origin) and marine water lead to suspect about some
Fig. IV Main fractionation steps and typical values of oxygen isotope composition (δ18O) in a temperate climate. δ18OSMOW,
standard mean δ18O in ocean water; δ18OV, δ18OP, δ18OLW, δ18O in water vapour, precipitation (either rainfall or snow) and leaf
water, respectively; VPD, vapour pressure deficit; gs, stomatal conductance. Original data from IAEA/WMO (2001) and Saurer
et al. (1997b).
25
Introduction
fractionation at the plant level. Gonfiantini et al. (1965) proved for the first time that the isotopic
composition of leaf water was enriched in heavy isotopes during transpiration. When plant roots take soil
water there is no fractionation, so the critical site of fractionation is the leaf. Summarising, the δ18O of
plant tissues reflects the variation in (1) δ18O in source water, (2) evaporative enrichment of leaf water
due to transpiration, and (3) biochemical fractionation during the synthesis of organic matter (Farquhar
and Lloyd 1993; Yakir 1992). The level of enrichment of leaf water above source water (∆18Oe) has been
modelled as follows (Dongmann et al. 1974; Farquhar and Lloyd 1993):
Eq. V
∆18Oe =ε*+ε k +(∆18Ov -ε k ) × (ea /ei )
where ε* is the proportional depression of vapour pressure by the heavier H218O, εk is the diffusion
fractionation through stomata and leaf boundary layer, ea and ei stand for vapour pressure in atmosphere
and intercellular space, respectively, and ∆18Ov is the oxygen isotope composition of water vapour in the
atmosphere (relative to source water). At constant temperature, and where source water and atmospheric
vapour have the same isotopic signature, ∆18Oe is linearly dependent on 1- ea/ei (Barbour et al. 2001).
According to this model, plants growing at higher humidity (i.e. higher ea) are expected to have lower
∆18Oe. Consequently, within the same environmental conditions, plants with higher stomatal conductance
(i.e. lower leaf temperature, which reduces ei) are also expected to show smaller ∆18Oe. This models
explains the isotopic enrichment of water at the site of evaporation, however, backward diffusion of the
enrichment is opposed by the convection of isotopically lighter source water to the sites of evaporation,
what is known as the Péclet effect (Barbour et al. 2004; Barbour and Farquhar 2000). The δ18O of leaf
water is, therefore, less depleted than that at the site of evaporation, so Eq. V overestimates the net effect
of transpirative enrichment. Leaf water enrichment is passed on to the organic molecules formed in the
leaf by exchange of oxygen atoms between carbonyl groups and water (Sternberg et al. 1986). The δ18O
of sucrose formed in the leaf is proportional to the signal of leaf water, although with 27‰ enrichment.
However, in the case of stem cellulose, most of the enrichment signal (derived from sucrose) is further
exchangeable with xylem water during the heterotrophic pathways of cellulose biosynthesis (Roden et al.
2000; Sternberg et al. 2003). This is particularly important for tree-ring studies, as will be discussed later
(section III.1.7).
26

Introduction
III. Palaeenvironmental studies
Reconstruction of past climate is necessary to understand the context associated to some of the most
intriguing natural processes: the evolution of species, their distribution or the occurrence of mass
extinctions. The same is valid at the human sphere, as some crucial changes in the history of humanity
(e.g. the onset of agriculture) cannot be dissociated from their environmental background. Moreover, a
better understanding of past climate changes would be valuable to determine to what extent current global
changes are unusual, as well as to improve long-term predictive models.
III.1. How can we study past climates?
Due to the scarcity of instrumental climate records prior to the XXth century, estimates of global
climate variability during past centuries must rely upon indirect “proxy” indicators. A proxy indicator is a
local record (natural or documentary) that is interpreted using physical or biophysical principles to
represent some combination of climate- or environmental-related variations back in time.
Palaeoenvironmental proxies have the potential to provide evidence for large-scale climatic changes prior
to the existence of instrumental or historical documentary records. Careful calibration and crossvalidation procedures are necessary to establish a reliable relationship between a proxy indicator and the
climatic variable or variables it is assumed to represent, providing a “transfer” function through which
past climatic conditions can be estimated. High-resolution proxy climate indicators, including tree rings,
corals, ice cores, and laminated lake/ocean sediments, can be used to provide detailed information on
climate variations back in time, which can be complemented by certain coarser resolution proxy
information (e.g. boreholes and non-laminated ocean sediment records). Some of the most commonly
used proxy indicators are outlined below, paying special attention on tree-rings, due to their relative
importance in the context of this Thesis.
III.1.1. Historical climatology
Historical climatology is based on the study of different kinds of references that can be associated to
climate events. For some areas, written evidences are available for several millennia and, although an
explicit record of climate variables is not usual, they are often referred indirectly. Thus, references to the
effects of flood events, long periods of drought or extremely cold winters can be found in religious
archives in the form of rogation ceremonies, as well as in agricultural records (Barriendos and MartínVide 1998; Bell 1975; Pfister et al. 1999).
III.1.2. Ice-cores
Ice cores from polar regions and mountain glaciers provide several indicators of past climates,
including stable isotopes (δ18O), the fraction of melting ice, accumulation rates, concentration of different
salts and acids, or the amounts of pollen or trace gases (e.g. CH4 or CO2) in the atmosphere, among others
(Alley et al. 1997; Francey et al. 1999). Ice core data has a high temporal resolution (from annual to
27
Introduction
seasonal), and provides simultaneous information from climate-related and atmospheric variables, that
might be helpful to assess the effect of CO2 increase in global climate. The best dated series are based on
sub-annual sampling of cores and the counting of seasonal ice layers, diving absolute errors of just a few
years in a millennium (Folland et al. 2001). Ice cores have the disadvantage of being located far from the
areas of human devolopment. This is a strength from the point of view of global climate issues, as they
are less affected by local anthropogenic effects. However, it is also a disadvantage in order to assess the
role of past climate changes in human evolution.
III.1.3. Pollen records
The particular biochemical composition of pollen grains makes them relatively resistant to chemical,
biological and physical damage. Consequently, pollen grains may survive millions of years in a large
variety of sedimentary environments: peat bogs, lake and marine beds, several kinds of loose terrestrial
sediments, and even in consolidated rocks (for example in stalagmites). As pollen deposition, for a given
species, is expected to be proportional to its abundance, palynologists are able to reconstruct past
vegetation from the study of fossil pollen assemblages. Furthermore, and provided a good knowledge of
the ecological range of a given taxa, functional group or plant community, it has been possible to derive
climatic information from pollen data (Lebreton et al. 2004; López-Sáez et al. 2003). Nevertheless, it
should be noted that anthropogenic influence on vegetation has increased exponentially since the onset of
agriculture. Thus, during great part of the Holocene, pollen data might have too human-derived noise to
provide reliable climatic information, although still giving useful information about the landscape
(Committee on abrupt climate change 2002; Lebreton et al. 2004). Indeed, most pollen assemblages for
the last two millennia reveal clear symptoms of anthropogenic disturbance, such as the abundance of
pollen from cereals and other crops, and the spread of ruderal species (Davis 1994; Lebreton et al. 2004;
López-Sáez et al. 2003).
III.1.4. Lake and ocean sediments
Annually laminated (varved) lake sediments also provide high-resolution records of palaeoenvironment. When annual deposition of varves can be independently confirmed through other dating
techniques (e.g. radiocarbon) they provide seasonal to inter-annual data. Three main climate variables
may influence lake varves: summer temperature, winter snowfall and rainfall. Moreover to
sedimentological studies (grain size, sedimentation patterns), lake sediments can be analysed from a
variety of approaches: isotope analyses (δ13C in organic matter, as a reflect of the dominant biota; δ18O in
carbonates and shells as indicator of evaporation rates), chemical analyses (changes in salt composition,
abundance of different organic compounds, trace elements), pollen records, diatoms analyses (Davis
1994; Riera et al. 2004; Rodó et al. 2002).
Ocean sediments also offer high-resolution archives of climate, applying similar methods as those
used in lake sediments. However, annually laminated sediments are not usual (Folland et al. 2001).
Otherwise, sedimentation rates may still be enough to provide information on a century or millennial
scale (Duplessy 2004; Moreno et al. 2004), based on radiocarbon or other external dating, such as
28

Introduction
volcanic ash shards. The range of variables that can be determined in marine beds is similar to that of
continental lakes: sedimentology, stable isotopes in shells and organic matter, chemical analysis, pollen
records (Committee on abrupt climate change 2002; Folland et al. 2001).
III.1.5. Coral reefs
Annual growths of coral skeletons provide palaeoenvironmental information for tropical and subtropical oceans and atmosphere. For example, they have the potential to sample variations in regions
sensitive to El Niño Southern Oscillation (ENSO) which can be useful to resolve large-scale patterns of
climate (Folland et al. 2001). Accurate age estimates are possible for most sites using a combination of
annual variations in skeletal density and geochemical parameters. Palaeoenvironmental reconstructions
from corals rely mostly on geochemical variables, such as trace elements or stable isotopes (δ13C and
δ18O) (Juillet-Leclerc and Yiou 2002; Rimbu et al. 2003).
III.1.6. Borehole measurements
This is a relatively recent methodology, which attempts to provide a direct estimate of ground surface
temperatures under certain simplifying assumptions about the geothermal properties of the earth.
Although long-term temperature reconstructions have been made for the last two millennia (Bodri and
Dovenyi 2004), they are based on assumptions not fully confirmed in such a large-scale, and the temporal
resolution of borehole estimates decreases sharply back in time. Thus, borehole estimations of
temperature are probably most useful for climate reconstructions over the last five centuries (Folland et
al. 2001). Moreover, non-temperature related factors such as land-use changes make often difficult the
interpretation of borehole data.
III.1.7. Tree-rings
Since the early works of Douglass in 1914 (see (Robinson et al. 1990) for a historical review), treerings have been extensively used as palaeoenvironmental proxies. Currently, dendroclimatology is a welldeveloped subdiscipline of dendrochronology. It is based on the fact that tree growth is often limited by a
variety of environmental factors, which may vary depending on the area studied (Cook and Kairiukstis
1990; Schweingruber 1988). For example, in arid and semiarid areas precipitation is the main factor
limiting tree growth (Adams and Kolb 2004; Lev-Yadun et al. 1981; Serre 1976), whereas in colder
regions temperature becomes the most limiting factor (Briffa et al. 2004; Panyushkina et al. 2003). As the
objective is to maximise the effect of a given climate variable on tree growth, that is, to obtain a sensitive
tree-ring series, the most useful trees are those found near the margins of their natural growth range
(ecotones) where a clear limiting factor can be identificable. Indeed, over an altitudinal transect, for
example, we may found significant differences in growth response (Adams and Kolb 2004; Gutierrez
1991; Tardif et al. 2003). As a rule, relatively isolated trees (i.e. without competition) but potentially
limited by the desired factor, are the material of choice. For example, if we wish to obtain good inferences
on precipitation, trees placed on slopes are preferable than those from the bottom of a valley.
29
Introduction
Growth Index
From individual tree-ring width series to large-scale chronologies
Tree growth is not only determined by climate, but is also affected by other, non-climatic factors.
Tipically, ring width decreases exponentially with tree age, according to the geometry of an increasing
trunk diameter (Kiviste et al. 2002; Rodriguez et al. 2003). However, competition and disturbance effects,
among others, might alter this general trend. For example, growth rate of an individual tree can be greatly
enhanced after a neighbour tree is cut. Thus, for environmental studies, this “undesired” variability should
be statistically removed through standardisation. This involves fitting a curve to the tree-ring series, and
then dividing each ring width value by the corresponding curve value. In an ideal tree, fitting an
exponential curve would be enough, but in most cases a second detrending is performed through a cubic
smoothing spline (Cook and Peters 1981). The spline acts as a high-pass filter, and is defined by a cut-off
wavelength. For example, if this wavelength is 20 years, it will remove 50% of variance at this
wavelength, with increasing percentages at longer wavelengths (97.5% at 50 years), and lesser removal at
shorter wavelengths (2.5% at 8 years). In some cases, further detrending is performed through
autoregressive models (Monserud 1986). The final result is always a series of indices with a long-term
mean of unity, where higher or lower values for a given year represent proportionally higher or lower
growths. To maximise the climate signal, the series obtained from several trees per site are averaged
(preferably using a bi-weight robust mean estimation, (Cook and Holmes 1986) to build up a site
chronology (Fig. V). The main handicap of statistical treatments is the risk of removing low-frequency
Year
A)
B)
Fig. V Example to illustrate the construction of a tree-ring chronology. Tree-ring cores of Pinus halepensis Mill., collected in a
North-facing slope near Ribaroja d’Ebre (Ferrio and Voltas, unpublished results). A) visual cross-dating of cores from different
trees; B) standardised chronology using a double detrending (exponential decay and spline smoothing with a cut-off wavelength
of 25 years).
30

Introduction
climate variability (Cook et al. 1995). In any case, the parameters of standardisation should be carefully
determined, always bearing in mind the aim of our particular study.
To end, it should be noted that the time-span of tree-ring chronologies is not limited to the oldest
living tree. Matching patterns in ring widths or other ring characteristics (such as ring density patterns)
among several tree-ring series allow the identification of the exact year in which each tree ring was
formed (cross-dating). This has allowed to construct long tree-ring chronologies, by overlapping extant
ring width series with those obtained from old buildings, fallen logs, submerged trunks and other kinds of
sub-fossil material. In Northern and Central Europe, pine and larch chronologies now extend more than
7000 years, and oak chronology extend over 9000 years, and attempts have been made to expand these up
to 12000 years by linking the oak chronology with “floating” pine chronologies (Becker et al. 1991;
Briffa and Matthews 2002; Friedrich et al. 2004; Pilcher et al. 1984).
Stable isotopes in tree-rings
By the time it was first suggested the presence of a climatic signal in plant stable isotopes, tree-ring
width analyses were being widely used in palaeoecological studies (Ehleringer and Rundel 1988;
Robinson et al. 1990). Hence, the technique was rapidly implemented in tree-rings and several early
studies on the relationship between stable isotopes and climate were performed on this material (Craig
1954; Epstein 1979; Gray and Thompson 1976; Libby et al. 1976). Since then, wood or cellulose isotopic
composition of different elements has been extensively used in palaeoenvironmental and ecological
studies (for further review see McCarroll and Loader 2004; Switsur and Waterhouse 1998; Warren et al.
2001).
Initially, variations in the δ13C of tree-rings was related to temperature (Stuiver and Braziunas 1987;
Wilson and Grinsted 1977) and later studies have also reported positive relationships with this variable
(Anderson et al. 1998; Saurer et al. 1995). However, according to current models of plant carbon
discrimination (see section II.2), little or no direct effect of temperature on δ13C is expected. In fact, most
of these studies found stronger relationships with other variables, such as relative humidity (Stuiver and
Braziunas 1987) or precipitation (Anderson et al. 1998; Saurer et al. 1995). Hence, these results are
probably derived from indirect relationships between temperature and plant water status. δ13C in treerings has shown to be strongly correlated to modelled soil water balance, precipitation and evaporative
demand (Dupouey et al. 1993; Korol et al. 1999; Saurer et al. 1997c; Warren et al. 2001). However, such
relationships appear to be restricted to seasonally dry climates: in other contexts, irradiance, altitude and
nutrient availability appear to be the main responsive of isotopic variations (Hultine and Marshall 2000;
Leavitt and Long 1991; Livingston et al. 1999; Warren et al. 2001).
Early studies on δ18O and δ2H in tree-rings found that they were strongly correlated with average
temperatures, which was initially attributed to the isotopic fractionation in precipitation (Epstein 1979;
Gray and Thompson 1976; Libby et al. 1976). However, more detailed physiological studies revealed that
the variability associated to evaporative enrichment could not be neglected (Dongmann et al. 1974;
Farquhar and Lloyd 1993). Thus, the observed correlations with temperature should be attributed to the
31
Introduction
synergistic effect of this variable on the isotopic signal in precipitation, and on evaporative enrichment
(McCarroll and Loader 2004). On the other hand, evaporative enrichment also explains the strong effect
of relative humidity on δ18O and δ2H reported by several authors (Burk and Stuiver 1981; Saurer et al.
1997a). Nowadays, δ18O and δ2H signatures in tree rings are known to be determined by three main
variables (Roden et al. 2000; Sternberg et al. 2003): the isotopic signature of source water, leaf
evaporative enrichment and exchange with xylem water during cellulose and/or lignin synthesis. The
latter is determinant for the observed relationship between δ18O of tree-ring cellulose and meteoric water,
as it enhances the source-water signal, softening the effect of leaf-level enrichment (Barbour et al. 2001;
Saurer et al. 1997b; Sternberg et al. 2003).
Although most of the works cited so far were limited to the last 100 or 200 years, isotope studies in
tree-ring have been also expanded over some large-scale tree-ring chronologies, providing high resolution
climatic reconstructions throughout the Holocene (Becker et al. 1991; Feng and Epstein 1994; Kromer et
al. 2004; Libby et al. 1976; McCornac et al. 1994).
III.2. Holocene climate variability
After the cold-dry period known as Younger Dryas (ca. 12,800-11,500 cal. BP), begins the longest
warm and stable period in the last 400,000 years, which defines the Holocene (Committee on abrupt
climate change 2002; Folland et al. 2001). This period coincides with the epoch of greatest human
development and, for some authors, was a crucial factor in the onset and spread of agriculture and modern
civilisation. However, even being of lower magnitude than the preceding ones, a detailed study of
Holocene climate reveals important climatic variations (Fig. VI). Between 11,500-10,500 cal. BP climate
was still cooler than present, while from 9,000 to 8,200 cal. BP it became wetter and warmer than present.
This was followed by a short cold period of about 200 years, around 8,200 cal. BP (Alley et al. 1997).
Between 8,000 and 4,500 cal. BP climate was somewhat warmer and wetter than today, reaching since
then environmental conditions relatively similar to those found in present times, except for some cold
phases (900-300 BCE, Iron Age Cold Epoch; 1,600-1800 CE, Little Ice Age) (Gribbin and Lamb 1978;
Van-Geel et al. 1998) and warm episodes (900-1400 CE, Medieval Warm Period) (Bradley et al. 2001;
Gribbin and Lamb 1978).
Nevertheless, the implications of these global climate reconstructions can be very variable at the
regional or local scale. In particular, precipitation regimes show a considerably spatial heterogeneity in
response to global climate phenomenons, as is the case for the North Atlantic Oscillation (NAO) or the
ENSO (Muñoz-Díaz and Rodrigo 2003; Rodó et al. 1997). The heterogeneous response of precipitation
against global climate changes is mostly due to the multiplicity of factors that determine precipitation:
circulation of air masses, orography, ocean temperature. This heterogeneity is not only evident from
current meteorological data, but also from palaeoenvironmental registers. Thus, for example, the cold
period occurring 8,000 years ago reveals a wetter/colder climate in NE Iberian Peninsula (Pérez-Obiol
and Julià 1994), but it was drier in the South of Spain (Magny et al. 2003), as well as in the Near East
(Van Zeist and Bottema 1988; Willcox 1999; Willcox 2002).
32

Introduction
At the regional scale, climatic reconstructions of high temporal resolution are already available for the
Iberian Peninsula. Most of these studies are based on the reconstruction of past vegetation, either from
palinology (Jalut et al. 2000; López-Sáez et al. 2003; Pérez-Obiol and Julià 1994) or from charcoal
studies (Allué 2002; Badal 1990). The study of vegetation, however, has the disadvantage of reflecting
both climate and human effects, which reduces its palaeoclimatic significance after the onset of
agriculture (Lebreton et al. 2004). Another important source of information is the reconstruction of lake
levels, based on a combination of stratigraphic and geochemical methods (Riera et al. 2004; Rodó et al.
2002), which has provided useful data on temperature and water availability. These studies are
complemented by the diverse techniques used to reconstruct marine temperature, in the Atlantic Ocean
and in the Mediterranean Sea, as sea temperature has a strong influence on continental climate (see e.g.
Moreno et al. 2004; Nebout et al. 2002). For the last millennium, these data has been complemented by
other approaches, such as dendrochronology (Creus and Saz 1999; Manrique-Menéndez and FernándezCancio 2000; Richter and Eckstein 1990) or documentary studies (Barriendos 1997; Barriendos and
Martín-Vide 1998).
Fig. VI Climate changes in central Greenland over the last 17,000 years, at 100 years intervals. Reconstructions of temperature
(black line) and snow accumulation rate (white line). Main climate events are highlighted: 1) Little Ice Age; 2) Medieval Warm
Period; 3) Iron Age Cold Epoch; 4) 8k2 episode; 5) Younger Dryas. Adapted from Committee on abrupt climate change (2002).
33
Introduction
IV. Archaeobotany
In the process of excavation of archaeological sites one comes across plant matter of one kind or
another. Most archaeological plant remains are found in a charred state and are often incomplete for
standard systemathic studies. In the past, these botanical remains received very scant attention. Although
some early studies date from the end of the XIXth century, the interest on plant remains did not began to
generalise until the works of Helbaek (Helbaek and Schultze 1981) in the Near East. Since then, and due
to its particular relevance for the origin of Old World agriculture, the Near East is still the region more
extensively studied from archaeobotany (see e.g. Bar-Yosef and Kislev 1986; Hillman 1984; Renfrew
1973; Van Zeist and Casparie 1984; Willcox 1995). In the Iberian Peninsula, the earliest archaeobotanical
works are those from Siret and Siret (1890), but the great impulse to the discipline came from Téllez and
Ciferri (1954) and, in the 60’s and 70’s, from M. Hopf (Zohary and Hopf 1988). In the 80’s, the extensive
work of Buxó (1997) helped to consolidate archaeobotanical studies in the area, that are now well
developed around the Iberian Peninsula (Allué 2002; Alonso 1999; Peña-Chocarro 1992; Ros 1997).
IV.1. Main approaches in archaeobotany
At the beginning, the interest of archaeobotany was centred on the origin of cultivated plants, from a
genetic point of view, as well as on the processes associated to plant domestication and agriculture. Later,
as the number of data available increased and new techniques became available, the initial interest on
taxonomy per se derived into the study of the complex interactions between human communities and
plants. This change in direction was evidenced through the foundation of the International Work Group of
Palaeoethnobotany (IWGP) in 1968, which has served as a productive discussion forum for European
archaeobotanists. Nowadays, the interest of archaeobotanists includes both the inference of plant
exploitation strategies in the past and the reconstruction of palaeovegetation. For the latter, it is of special
relevance a subdiscipline of archaeobotany, the anthracology or charcoal analysis, which is devoted to the
study of wood remains. This approach has been well developed in Europe through the work of Vernet and
his numerous co-workers and disciples (Bazile et al. 1977; Ros 1997; Vernet and Thiebault 1987).
Nevertheless, the use of charcoal analysis to reconstruct vegetation is rather controversial, as it attempts
to derive vegetation from material that has been collected by human groups, thus assuming that this
would reflect plant communities. However, charcoal remains from archaeological contexts reflect the
interaction between vegetation and their use by human groups. Some charcoal studies are indeed focused
on the socio-economic (e.g. fuel exploitation, collection strategies) implications of this kind of remains
(Piqué 1998; Willcox 1999). Thus, plant macroremains might provide also useful information from a
palaeoenvironmental point of view, but the interpretation of results require a careful consideration of
other factors determining the composition of plant assemblages.
34

Introduction
IV.2. Some methodological aspects
IV.2.1. Preservation of fossil plant remains
In temperate climates, the most common way of preservation of plant remains is carbonisation. This is
the case of wood charcoal, either derived from occasional fires or from the use of wood as fuel. Seeds and
grains can also be burnt during different phases of food preparation (e.g. toasting), or included within
animal dung used as fuel (Buxó 1997; Van Zeist and Casparie 1984). When plant materials are combusted
under poor oxygen availability, they become carbonised, which prevents decomposition of plant remains
by fungal or bacterial decay. However, as carbonisation increases, plant remains become more brittle, and
thus the final preservation requires the ability to survive subsequent physical constraints during burial and
recovery (Colledge 2001; Wright 2003). Plant remains can be also preserved in the form of mineralised
tissues. In some cases, plant material may be preserved in an uncharred, dry, or saturated state. In very
arid and desert climates, like ancient Egypt or coastal Peru, dried plant parts may last for millennia. Even
in temperate climates, plants may be well preserved in dry caves and rockshelters. Saturated sites (bogs,
swamps, lakes, etc) may also preserve fragile plant materials indefinitely, and this has been profited, for
example, to build long tree-ring chronologies in Northern and Central Europe (McCarroll and Loader
2004).
IV.2.2. Recovering of plant remains
Until relatively recently, most plant remains recovered from archaeological context were detected by
chance. If a large quantity of charred seeds, or a big piece of wood, caught the eye of the archaeologist,
this material would be gathered up and saved, otherwise not. However, small charred seeds and plant
parts are not easily detected by the naked eye and, as a consequence, specialized means are necessary to
obtain such remains. The most common method by which these smaller macrobotanical plant fragments
are concentrated and recovered is through the technique known as flotation (see Fig. VII). Many different
flotation systems have been devised, but all of them depend upon the same basic principle: if
archaeological sediment is released into a container filled with water, then the sediment sinks and the
charred plant remains float. There are several techniques of flotation (Buxó 1997): some techniques are
multiple-operator, machine-assisted assembly lines, whereas others are simple, one-person bucket or
barrel operations. It is sufficient to note here, however, that all of these systems are aimed at retrieving
charred macrobotanical plant remains, and that the main purpose is the same for all: to concentrate and
collect charcoal that is dispersed throughout archaeological deposits.
Although the use of flotation has found worldwide acceptance, not all archaeologists employ it as a
technique to retrieve plant remains. In some areas, archaeological sediments may not be amenable to
flotation. Even in cases where flotation is not physically possible, however, attempts are often made to
recover plant remains. Dry or water sieving of sediments, for example, is routinely used in some
European countries to obtain plant remains. On the other hand, the recovery and study of
archaeobotanical remains is time-consuming and expensive and thus many archaeological projects simply
do not have the necessary time and money. Fortunately, the interest on archaeobotanical studies is going
beyond traditional archaeology, thus making accessible alternative sources for funding.
35
Introduction
Fig. VII Examples of the main steps in the recovery of archaeobotanical remains. On the left, (top) partial view of the exacavated
strata in Tell Halula, and (bottom) detail of charcoal remains included in the sediment. On the right, (top) extraction from the
sediment of charcoal and charred seeds by flotation, and (bottom) archaeological grains of wheat and barley. Original pictures
from J.L. Araus and R. Buxó.
IV.2.3. Dating of samples
The dating of archaeobotanical remains is not a single issue. The radiocarbon dating method, based on
the rate of decay of the unstable isotope 14C, is today the most widely applied dating technique for the late
Pleistocene and Holocene periods (Taylor and Aitken 1997). 14C is formed in the upper atmosphere
through the effect of cosmic ray neutrons upon 14N. It is rapidly oxidised and transformed into 14CO2, and
subsequently fixed by plants. As soon as the plant dies, it ceases 14CO2 uptake, and 14C content in plant
tissue decays progressively. In 1949, Libby, Anderson and Arnold were the first to measure the rate of
this decay. They found that after 5568 years, half the 14C in the original sample will have decayed and
after another 5568 years, half of that remaining material will have decayed, and so on. The half-life is the
name given to this value, which Libby measured at 5568±30 years. Currently, this value is considered to
be 5730±40 years (“Cambridge” half-life). The initial solid carbon method developed by Libby and his
collaborators was replaced with the Gas counting method in the 1950's. Liquid scintillation counting
(LSC), utilising benzene, acetylene, ethanol, methanol etc, was developed at about the same time. Today
the vast majority of radiocarbon laboratories use these two methods of radiocarbon dating. Their main
36

Introduction
handicap is the amount of sample required: even using small sample capabilities, about 100 mg are
required for moderate precision datings. Of major recent interest is the development of the Accelerator
Mass Spectrometry method of direct C14 isotope counting, which allows milligram sized samples to be
dated. Radiocarbon dating should be calibrated externally to account for past changes in atmospheric 14C
composition, and the final accuracy varies from several hundred years to a few decades, depending on the
period considered (Jull 2005; Stuiver et al. 1998). On the other hand, the elevated cost of these analyses
strongly limits the amount of samples to be dated. Consequently, most plant macroremains are assigned
an age according to the dating obtained from other charcoal, seeds or bones from the same (or the nearest)
stratigraphic unit. In some cases, a combination of stratigraphic and archaeological methods (e.g. based
on ceramics and other artefacts) can provide more accurate dating than radiocarbon analyses. Dating of a
given stratigraphic unit from wood charcoal has an additional uncertainty, as a given wood fragment
might be older than the sediment in which is included. Thus, seeds and other short-living plant remains
are often preferred for dating.
IV.3. Stable isotopes in archaeobotanical remains
As discussed in sections II and III, stable isotopes in plant tissues reflect the environmental conditions
in which they were developed. Charred archaeobotanical remains are indeed a subproduct of plant tissues,
derived form their partial combustion, and thus may preserve some kind of environmental signal in their
isotopic composition. De Niro and coworkers (DeNiro and Hastorf 1985; Marino and DeNiro 1987) were
the first to propose the analyses of δ13C and δ15N in archaeobotanical remains, with the aim to provide
reference values for dietary studies. They performed experimental carbonisations of several types of
seeds, and assayed different treatments to remove soil contaminations, such as carbonates or humic acids,
finding little change in δ13C due to carbonisation and chemical treatments, being greater for δ15N. Such an
extensive work, along with the increasing physiological knowledge about δ13C variability in grain crops
(Condon et al. 1987; Farquhar et al. 1982; Farquhar 1984), provided the necessary background for the
first attempts to apply δ13C in archaeological grains to infer the water status of ancient crops (Araus et al.
1997a; Araus et al. 1999a; Araus and Buxó 1993). In these works, Araus and his colleagues developed
quantitative models to estimate past water inputs in winter cereals (wheat and barley) through the analysis
of δ13C in grains. Following a similar approach, and based on the close relationship between δ13C (as
indicator of water status) and cereal yield (Araus et al. 1998; Condon et al. 1987; Voltas et al. 1998), they
used δ13C of archaeological grains to infer ancient crop yields (Araus et al. 1999b; Araus et al. 2001;
Araus et al. 2003).
From the point of view of climatic reconstruction, the analysis of crop remains has the disadvantage
they may reflect either climatic or agronomic changes affecting crop water status (Araus et al. 1997b).
However, provided an independent estimation of climate for the archaeological sites studied, this
“handicap” might become an useful tool to reconstruct ancient agronomic practices. In this context, the
use of charcoal remains of forest species appears to be the best alternative. The only studies available on
the δ13C in ancient wood charcoal are those from February and Van der Merwe (1992) and Vernet et al.
37
Introduction
(1996). Mostly based on the comparison with other palaeoenvironmental evidences, both concluded that
past changes in the δ13C of charcoal could be related to changes in water availability, as occurred in intact
wood (see section III.1.7). However, they did not studied the potential effect of carbonisation on wood
δ13C, which, according to previous works, might be considerable (Florit 2001; Jones and Chaloner 1991).
Consequently, prior to the application of stable isotopes in wood charcoal for palaeoenvironmetal studies,
it is necessary to assess the effects of carbonisation under different conditions on the original
environmental signal of wood.
38

Introduction
V. Objectives
As already stated in the overview, the general objective of this Thesis is to develop new tools in order to
get insight into the climatic and agronomic conditions prevailing in ancient agriculture. Due to the
interdisciplinary nature of this work, we have grouped the objectives into different topics:
I. Reconstruction of climate
Ecophysiological studies on extant trees
I.1 To compare the dependency of tree-ring growth and δ13C on water availability for two
Mediterranean species with different physiological strategies to cope with drought: the partially
drought-tolerant Quercus ilex L., and the drought avoiding, P. halepensis Mill. We aim to assess
our hypothesis that a drought-tolerant species, such as Q. ilex would show lower increase in
Water Use Efficiency (WUEi) under drier conditions than a drought-avoiding species (e.g. P.
halepensis).
I.2
To determine which are the environmental variables (temperature, precipitation, vapour
pressure deficit) determining δ18O variation in whole wood and its main components
(holocellulose and lignin) in a drought avoiding species, such as P. halepensis. We aim to test
our hypothesis that, in a species with a strong stomatal regulation, the signal of source water
(δ18OR) would be lost due to the great variability in evaporative enrichment at the leaf level.
Climatic inferences from modern wood
I.3 To determine to what extent different climatic variables (precipitation, temperature, evaporative
demand) are reflected in wood δ13C in Q. ilex and P. halepensis, in order to use δ13C as a
(palaeo)environmental proxy.
I.4
To assess the need for cellulose purification in P. halepensis in order to retrieve a climatic
signal from δ13C and δ18O in wood. Cellulose purification is not possible in wood charcoal, so
the lack of a climatic signal in lignin would preclude its use as a (palaeo)environmental proxy.
I.5
To assess, in P. halepensis, the potential complementarity of δ13C and δ18O analyses for
(palaeo)environmental studies, as both are expected to respond in some way to stomatal
processes.
39
Introduction
Stable isotopes in fossil charcoal and palaeoclimate
I.6 To assess the effect of carbonisation on the δ13C of P. halepensis wood. In case of a significant
effect, to develop a model to correct δ13C shifts due to carbonisation.
I.7
To quantify changes in aridity in the Cinca and Segre Valleys (Ebro Depression) during the last
four millennia, from the analysis of δ13C in wood charcoal.
II. Reconstruction of crop conditions
II.1 To update current models adressed to determine past water status in naked wheat (Triticum
aestivum/durum) and hulled barley (Hordeum vulgare), and to apply them to ancient crops
cultivated in NE Iberian Peninsula (from ca. 5,800 BCE to XIth century), SE Iberian Peninsula
(ca. 4,500-300 BCE), as well as in the Fertile Crescent (ca. 7,900-6,500 BCE).
II.2 To explore new strategies to deal with δ13C data on ancient crops, by comparing objectively the
observed trends in different crop species, e.g. between cereals and legumes, or between wheat
and barley. Specifically, we aim to find evidences that wheat was already favoured by ancient
farmers, sowing it in better soils than barley, or under supplementary irrigation, and thus having
better water status.
II.3 To develop a model to quantify original grain weight from the dimensions of charred cereal
(wheat and barley) grains, to facilitate the comparison with modern agronomic data. Using this
model, to assess the evolution of grain weight in cereals (wheat and barley) from the Cinca and
Segre Valleys during the last four millennia. To find out potential reasons for the observed
trends, by relating grain weight with estimations of crop water availability (through δ13C of
grains) and estimated precipitation (through δ13C of charcoal).
II.4 By comparing observed trends in water availability for cereals in NW Mediterranean with those
from fossil charcoal, to determine to what extent they can be attributed to climate or agronomic
practices.
40

Introduction
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48

Chapter 1
“Brotas derecha o torcida
con esa humildad que cede
sólo a la ley de la vida,
que es vivir como se puede.”
Antonio Machado. Las encinas
49
Chapter 1
J.P. FERRIO1 • A. FLORIT1 • A. VEGA1 • L. SERRANO1 • J. VOLTAS1*
∆13C and tree-ring width reflect different drought responses in
Quercus ilex and Pinus halepensis
1
Departament de Producció Vegetal i Ciència Forestal, E.T.S.E.A - Universitat de Lleida,
Av. Rovira Roure 191, Lleida E-25198, Spain
*Corresponding author; e-mail: [email protected]
Abstract Holm oak (Quercus ilex L.) and Aleppo pine (Pinus halepensis Mill) are representative of
two different functional types of trees extensively found in the Mediterranean: evergreen
sclerophyllous and drought-adapted conifers. The former is considered a partially drought-tolerant
species, whereas the latter is a typically drought-avoiding, water-saving species. We postulated that
contrasting strategies in response to water deficits in Q. ilex and P. halepensis would lead to a
differential sensitivity to changes in water availability. To test this hypothesis, we compared the
response of both species in growth rate (measured as radial increments) and intrinsic water use
efficiency (WUEi, as inferred from carbon isotope discrimination -∆13C- in wood samples) among
sites from different provenance regions in NE Spain. We found significant differences in ∆13C and
growth among provenance regions, partly explained by contrasting water availability. Wood ∆13C was
positively related with precipitation and the ratio between precipitation and potential
evapotranspiration (P/E). However, these relationships were stronger in P. halepensis (for P/E,
r2=0.67, p<0.001) than in Q. ilex (r2=0.42, p<0.01). In addition, radial growth was positively related
with precipitation and ∆13C in P. halepensis (r2=0.32 and r2=0.35, respectively, P<0.01), but not in Q.
ilex. We concluded that P. halepensis was more sensitive than Q. ilex to water availability, showing
faster increase in WUEi in response to water stress. We also found that the effect of North/South
aspect on ∆13C and growth was site-specific, and unrelated to climatic variables.
Keywords climate • carbon isotope discrimination • water use efficiency • sclerophyllous • conifer
Abbreviations ∆13C, Carbon isotope discrimination; P/E, ratio between precipitation and potential
evapotranspiration;WUEi, intrinsic water use efficiency
1.1. Introduction
Dominant tree species in Mediterranean
ecosystems are either evergreen sclerophyllous or
conifers adapted to the scarcity of water resources.
Holm oak (Quercus ilex L.) and Aleppo pine
(Pinus halepensis Mill) are representative of each
one of these functional types and both are
extensively found in the Mediterranean. However,
they show considerable differences not only on
their structure but also on their ecophysiological
responses to cope with drought. Q. ilex is a deeprooted species that does not depend only on rainfall
supplies but on a very effective water uptake
(Valentini et al. 1992). It is also able to withdraw
water from very dry soils, leading to the
maintenance of relatively high stomatal
conductances even during drought periods. Hence,
it should be regarded as a partially drought-tolerant
Published in Oecologia 137:512-518 (2003) 51
species (Damesin et al. 1998). In contrast, P.
halepensis is known to reduce drastically water use
during drought by stomatal closure (Borghetti et al.
1998). Its indeterminate free growth allows it to
cease growth during drought events, recovering
rapidly when water becomes available (Nicault et
al. 2001; Liphschitz and Lev-Yadun 1986).
Therefore, it can be considered as a true droughtavoiding species, fully adapted to effective water
saving.
Carbon isotope discrimination (∆13C) in C3
plant tissues constitutes an integrated record of the
ratio of intercellular to atmospheric concentration
of CO2 (ci/ca) during the period in which the
carbon was fixed, and thus reflects the balance
between
assimilation rate
and
stomatal
conductance (intrinsic water use efficiency,
WUEi). ∆13C has been related to different variables
associated with plant water stress, such as predawn
water potential (Damesin et al. 1998; Warren et al.
2001), climatic gradients of humidity (Saurer and
Siegenthaler 1989), precipitation (Korol et al.
1999; Schulze et al. 1998; Warren et al. 2001), and
other indicators of water availability (Dupouey et
al. 1993; Livingston et al. 1993). It may be
advisable, however, to complement ∆13C analyses
with other indicators of plant function, such as
morphological attributes (Damesin et al. 1997;
Fleck et al. 1996) or plant growth (Dupouey et al.
1993; Brooks et al. 1998) to improve the
understanding of the ecological meaning of ∆13C
values.
Information relating ∆13C and water availability
is still scarce for conifers from semiarid
environments (Williams and Ehleringer 1996) and
Mediterranean oaks (Damesin et al. 1998). At this
point, we hypothesised that contrasting strategies
in response to water deficits in Q. ilex and P.
halepensis would lead to a differential sensitivity
to changes in water availability, and that this could
be tracked by means of ∆13C analysis. To test this
hypothesis, we compared the response of the two
species in terms of radial growth rates and wood
∆13C over a wide range of sites differing in
precipitation regime. ∆13C would provide an
integrated record of WUEi during the growth
period, whereas radial growth would indicate the
effect of water shortage on the overall tree
functioning. As a secondary objective, we studied
the potential effect on ∆13C and growth of
increased evapotranspiration in South-faced slopes
by sampling in opposite slopes (North/South).
1.2. Materials and Methods
1.2.1. Plant material
We sampled wood cores of Aleppo pine (Pinus
halepensis Mill.) and holm oak (Quercus ilex L.)
from different sites in NE Spain. As sampling
strategy we used the current division in provenance
regions of the distribution area for both species in
Spain (Martín et al. 1998). A provenance region
defines an adaptive unit in which phenotypically or
genetically similar stands or seeds are found
(European Council directive 1999/105/EC). A total
of six provenance regions for P. halepensis and
Fig. 1.1 Geographical distribution of provenance regions of
Quercus ilex and Pinus halepensis included in this work (see
Table 1.1 for a detailed region description). Sampling sites
within each region are marked as points. Adapted from Martín
et al. (1998).
52
 Chapter 1. ∆13C and tree-ring width reflect different drought responses in Quercus ilex and Pinus halepensis
five provenance regions for Q. ilex were sampled
(Fig. 1.1, Table 1.1). Due to their relative inhomogeneity, regions 4 and 10 for Q. ilex are
divided into sub-regions (Martín et al. 1998),
which will be taken here as separate regions. We
selected
sampling
sites
with
available
meteorological records within a 10km radius and
representative of the environmental variability
within each provenance region (one to three sites
per region). Wood cores were extracted from the
south side of mature, dominant and healthy trees,
sampling four to six trees per aspect (North or
South) within each site. P. halepensis cores were 5
mm in diameter, whereas for Q. ilex we took 12
mm-cores
to allow for tree-ring dating, as
described by Gené et al. (1993).
1.2.2. Tree-ring dating
and sample preparation
Samples were oven-dried at 60ºC for 48h
before being polished to allow tree-ring dating. We
measured tree-ring widths with a binocular
microscope coupled to a PC with the program
TSAP v. 3.0 (Frank Rinn, Heidelberg). After visual
cross-dating, assisted with tree-ring measurements,
we selected a fragment of the cores corresponding
with the periods 1975-1999 and 1980-2001, for P.
halepensis and Q. ilex, respectively. We calculated
mean annual radial growth from tree-ring widths
within these periods as an indicator of long-term
secondary growth. The fragments from the same
site and aspect where then pooled and milled
Table 1.1 Main climatic parameters for the provenance regions of Quercus ilex and Pinus halepensis included in this work. Intersite range values shown between brackets.
Region
P (mm)
T (ºC)
Ratio P/E
Im
Köppen classification
0
1
-2
30
-7
-20
-22
-30
Cfa, Humid subtropical
Cfa, Humid subtropical
Cfa, Humid subtropical
Cfb, Marine
Csa, Pure mediterranean
Csb, Coast mediterranean
BSk, Cool semiarid
BSk, Cool semiarid
10
-19
-9
-13
-22
-27
Cfa, Humid subtropical
Csa, Pure mediterranean
Csa, Pure mediterranean
Csa, Pure mediterranean
Csa, Pure mediterranean
BSk, Cool semiarid
Quercus ilex L.
4a
4b
4c
5
6
10b
10c
F
620 (561-678)
637 (449-1013)
616 (536-701)
851 (642-1019)
553 (529-576)
401 (392-410)
375
372
14.1 (13.7-14.5)
13.5 (12.9-14.2)
13.1 (11.6-14.3)
12.8 (12.2-13.2)
13.8 (13.7-13.9)
11.6 (10.3-13.0)
10.3
15.3
0.8 (0.8-0.9)
0.9 (0.6-1.3)
0.8 (0.7-1.0)
1.2 (0.9-1.4)
0.7 (0.7-0.8)
0.6 (0.6-0.7)
0.6
0.5
Mean ± SD
623 ± 221
13.3 ± 1.4
0.9 ± 0.3
14.9 (14.1-15.4)
15.6 (15.2-15.9)
14.2 (12.7-15.1)
13.5 (12.9-14.0)
14.6 (13.2-15.9)
14.3 (13.5-14.9)
1.0 (0.9-1.1)
0.6 (0.6-0.6)
0.7 (0.5-1.0)
0.7 (0.6-0.8)
0.6 (0.4-0.8)
0.5 (0.5-0.6)
Pinus halepensis Mill.
1
744 (693-809)
2
493 (477-510)
3
566 (395-753)
4
497 (422-551)
5
450 (361-563)
6
385 (362-407)
Mean ± SD
523 ± 140
14.4 ± 1.1
0.7 ± 0.2
P, annual precipitation; T, mean temperature; P/E, ratio between
Im, Thornthwaite moisture index (Thornthwaite 1948; Köppen and Geiger 1936).
precipitation
and
potential
evaporation;
Published in Oecologia 137:512-518 (2003) 53
(IKA-A10) to a fine powder. We used intact wood
tissue for isotopic analyses, without any chemical
pre-treatment. The postulated need for cellulose
purification from wood samples to assess
environmental effects on ∆13C has been questioned
by many authors. Indeed, similar relationships can
be found using either whole wood or cellulose
(Warren et al. 2001; Korol et al. 1999).
1.2.3. Meteorological data
Meteorological data for both periods (19751999 for P. halepensis and 1980-2001 for Q. ilex)
were supplied by the Instituto Nacional de
Meteorología and the Confederación Hidrográfica
del Ebro. We took monthly values for precipitation
and mean temperature. From the temperature and
the
latitude,
we
calculated
potential
evapotranspiration following Thornthwaite (1948).
The ratio between precipitation and potential
evapotranspiration (P/E) was also calculated as an
index of water availability. Wherever the altitude
of the sampling site differed from that of the
meteorological station, we applied the following
correction (Gandullo 1994): for temperature,
decrease in 0.6ºC per 100m; for precipitation, 8%
increment precipitation is mostly convective, and
not related with altitude.
1.2.5. Statistical analysis
The data were subjected to analysis of variance
(ANOVA), with species, provenance region, site
nested to provenance region, and aspect included
as factors in the model. Covariance analyses were
performed to assess the influence of environmental
variables on the aforementioned factors.
Heterogeneity of slopes ANOVAs were performed
to detect differences between species in the
response of ∆13C and radial growth to
environmental variables (i.e. to test whether or not
the regression coefficients are constant over
groups, as assumed in a covariance analysis; Little
et al. 1991). Simple correlations and linear
regressions were calculated to assess relationships
between variables. Unless otherwise stated,
differences were considered statistically significant
when P < 0.05.
1.2.4. Carbon isotope discrimination
The 13C/12C ratios of wood samples were
determined by mass spectrometric analysis and
results expressed as isotopic composition (δ13C).
We calculated ∆13C from δa and δp (air and plant
δ13C), as defined by (Farquhar et al. 1982): ∆13C =
δa-δp/(1+δp/1000). We took mean values of δa=7.7‰ and δa=-7.8‰ for the periods 1975-1999 and
1980-2001, respectively, obtained from Francey et
al. (1999) and the CU-INSTAAR/NOAA-CMDL
network(ftp://ftp.cmdl.noaa.gov/ccg/co2c13/flask/r
eadme.html).
54
 Chapter 1. ∆13C and tree-ring width reflect different drought responses in Quercus ilex and Pinus halepensis
1.3. Results
1.3.1. ∆13C and growth for Quercus ilex
and Pinus halepensis
∆13C in Q. ilex varied among sites from 16.8‰
to 18.5‰, with a mean of 17.9‰. ∆13C was
generally lower for P. halepensis (mean of 16.5‰)
and showed greater range of variation (15.4‰17.6‰). Differences in ∆13C between species were
significant, even after removing the effect of
climatic variables (precipitation, temperature, P/E)
in a covariance analysis.
We also found significant differences in mean
annual radial growth between species. They ranged
from 0.3 to 1.1 mm year-1 for Q. ilex (mean= 0.7
mm year-1), whereas P. halepensis displayed
greater values, ranging from 0.9 to 3.9 mm year-1,
with a mean of 1.9 mm year-1.
1.3.2.Geographic variation
There were significant differences in ∆13C
among provenance regions for both species. For Q.
ilex we found the highest and lowest values for
∆13C in regions 5 (18.5‰), and F (16.8‰),
respectively (Fig. 1.2). For P. halepensis, mean
values of ∆13C ranged from 15.9‰ in region 6 to
17.3‰ in the region 1. Differences between
species were not detected for the North/South
aspect. As for ∆13C, radial growth showed
significant differences among provenance regions.
We did not detect significant differences in radial
growth due to North/South aspect, but there were
significant interactions between aspect and
provenance region for both species. Intra-site
North-South differences ranged from –0.25 to
+0.29 mm year-1 for Q. ilex, and from –0.39 to
+0.40 mm year-1 for P. halepensis. However, these
differences did not follow any clear pattern, being
unrelated with either ∆13C or any climatic variable
studied.
1.3.3. Relationship between climatic variables,
∆13C and growth
Relationship with annual means
Site means of ∆13C were positively correlated
with both annual precipitation and P/E for Q. ilex
and P. halepensis, although these relationships
were stronger for P. halepensis (Table 1.2). We
also found a negative association between ∆13C
and potential evapotranspiration for P. halepensis
(Table 1.2). By plotting the relationship between
precipitation (or P/E) and ∆13C, a trend towards
19.5
Wood ∆13C (‰)
19.0
(a) Quercus ilex
(b) Pinus halepensis
North
South
18.5
18.0
17.5
17.0
16.5
16.0
15.5
4a 4b 4c
5
6 10b 10c F
1
2
3
4
5
6
Provenance region
Fig. 1.2 Mean wood ∆13C in Q. ilex and P. halepensis trees grown in two slope aspects (North/South) within the provenance
regions described in Table 1.1. Error bars indicate the standard error of the means (N=1-3).
Published in Oecologia 137:512-518 (2003) 55
Table 1.2 Across-sites correlation coefficients between annual means of climatic variables and either carbon isotope
discrimination (∆13C, ‰) or annual radial growth (mm) of Quercus ilex and Pinus halepensis.
Quercus ilex (N=17)
Climatic variables
Wood ∆13C
Precipitation (P)
Mean temperature
Potential evapotranspiration (E)
Ratio P/E
* P<0.05; ** P<0.01; *** P<0.001
Pinus halepensis (N=18)
Radial growth
0.59**
-0.17
-0.30
0.63***
Wood ∆13C
Radial growth
0.73***
-0.30
-0.46*
0.77***
0.57***
0.27
0.13
0.53***
-0.11
-0.24
-0.28
-0.08
saturation was observed as water availability
increased (i.e. precipitation and P/E) (Fig. 1.3).
Thus, a log transformation fitted better the data. Q.
ilex ∆13C increased with the log of precipitation at
a rate of 0.97‰ per log unit, with a coefficient of
determination (r2) of 0.36 (Fig. 1.3a), whereas the
increase for P. halepensis was 1.91‰ per log unit
(r2=0.59). A log transformation for P/E gave
similar fits, with r2=0.42 and r2=0.67 for Q. ilex
and P. halepensis, respectively (Fig. 1.3b). We
detected strongly significant differences (P<0.001)
between species when comparing the slopes of the
relationship between log transformed data (either
precipitation or P/E) and ∆13C.
Site mean annual radial growth was not
associated with climatic variables for Q. ilex,
whereas it was positively related with precipitation
and P/E for P. halepensis (Table 1.2). Radial
growth of P. halepensis increased linearly with
annual precipitation at a lapse rate of 3.31 µm year1
mm-1 (Fig. 1.4a). Radial growth was also
positively related with ∆13C in P. halepensis (Fig.
1.4b), but not in Q. ilex.
Relationship with monthly means
Correlations between Q. ilex ∆13C and monthly
precipitation were generally higher in autumnwinter than in spring-summer months, of which
19.0
Wood ∆13C (‰)
18.5
18.0
17.5
17.0
16.5
Q. ilex
P. halep.
16.0
15.5
200
400
∆Qi=11.72+0.97*ln(P) r2=0.36**
∆Qi=18.10+1.04*ln(P/E) r2=0.42**
∆Ph= 4.63+1.91*ln(P) r2=0.59***
∆Ph=17.31+1.93*ln(P/E) r2=0.67***
600
800
1000 1200
Precipitation (mm)
0.4
0.6
0.8
1.0
1.2
1.4
1.6
Ratio P/E
Fig. 1.3 Relationship between wood ∆13C in Q. ilex and P. halepensis and either mean annual precipitation (P) or the ratio
between precipitation and potential evapotranspiration (P/E). ∆Qi and ∆Ph represent ∆13C in Q. ilex and P. halepensis,
respectively. Error bars indicate the standard error of the means (N=2). * P<0.05; ** P<0.01; *** P<0.001
56
 Chapter 1. ∆13C and tree-ring width reflect different drought responses in Quercus ilex and Pinus halepensis
only June showed a significant relationship (Fig.
1.5a). Conversely, Q. ilex ∆13C was not
significantly correlated with monthly values of
potential evapotranspiration, although some
negative trend was observed for summer months
(Fig. 1.5b). ∆13C of P. halepensis showed
significant correlations with monthly precipitation
throughout the year (Fig. 1.5c). We also found a
significant, negative relationship between P.
halepensis ∆13C and potential evapotranspiration,
but only during summer (from June to September,
see Fig. 1.5d).
Radial growth (mm)
4.5
-3
RGPh= 0.21+3.3*10 *P
There was a significant, negative relationship
between radial growth and September precipitation
for Q. ilex (Fig. 1.5a), whereas correlations
between
radial
growth
and
potential
evapotranspiration, although slightly negative
throughout the summer months, were still
not significant (Fig. 1.5b). Radial growth of
P. halepensis was significantly correlated with
monthly precipitation in March and from July to
December (Fig. 1.5c), being positively associated
with potential evapotranspiration in winter
(December-February, Fig. 1.5d).
2
r =0.32**
RGPh= -10.62+0.76*∆
2
r =0.35**
4.0
3.5
3.0
2.5
2.0
Q. ilex
P. halep.
1.5
1.0
0.5
0.0
200
400
600
800
1000
1200
15.5 16.0 16.5 17.0 17.5 18.0 18.5 19.0
Wood ∆ C(‰)
13
Precipitation (mm)
Correlation coefficients (r)
Fig. 1.4 Relationship between mean radial growth (RG) in Q. ilex and P. halepensis and either mean annual precipitation (P) or
wood ∆13C (∆). RGPh, radial growth in P. halepensis. Error bars indicate the standard error of the means (N=8-12). * P<0.05; **
P<0.01; *** P<0.001
0.8
(a) Precipitation (P)
(b) Potential evapotranspiration (E)
0.4
0.0
-0.4
-0.8
0.8
Q. ilex
Q. ilex
(c) Precipitation (P)
(d) Potential evapotranspiration (E)
0.4
0.0
-0.4
-0.8
Wood ∆13C
Radial growth
P. halepensis
P. halepensis
Ja Fe Mr Ap My Jn Jl Au Se Oc No De
Ja Fe Mr Ap My Jn Jl Au Se Oc No De
Fig. 1.5 Correlation coefficients between wood ∆ C / radial growth and either monthly precipitation (a, c) or monthly potential
evapotranspiration (b, d) for Q. ilex (a, b) and P. halepensis and (c, d). Dotted lines indicate the threshold-value for significant
correlations (P<0.05).
13
Published in Oecologia 137:512-518 (2003) 57
1.4. Discussion
1.4.1. ∆13C response to water availability
and climate
Wood ∆13C in Q. ilex and P. halepensis was
positively related with annual precipitation and the
ratio P/E, thus reflecting the effect of both climatic
variables on plant water status. Several authors
have reported similar relationships between
indicators of water availability and wood ∆13C,
either in conifers (Korol et al. 1999; Warren et al.
2001) or in broadleaf trees (Saurer et al. 1995;
Dupouey et al. 1993). Previous works in conifers
have shown that ∆13C is more sensitive to water
availability as water becomes more scarce,
reaching an asymptotic level around P/E>1 (Korol
et al. 1999; Warren et al. 2001). Although our
climatic range exceeds only slightly this threshold,
models greatly improved after log transformation
of the variables. Our results thus confirm that P/E
exerts an important control on ∆13C, but only when
water availability is the main limiting factor for
photosynthesis.
On the other hand, ∆13C was not significantly
related with temperature, although previous studies
placed temperature among the factors best
correlated with ∆13C (Stuiver and Braziunas 1987;
Anderson et al. 1998; Brooks et al. 1998). The
relatively narrow range of temperatures (10.314.5ºC for Q. ilex and 12.7-15.9ºC for P.
halepensis), as well as the absence of cold climates
in this range, might explain the lack of temperature
effect on ∆13C. Nevertheless, a mechanistic
explanation for a negative relationship between
temperature and ∆13C still lacks, and often it can be
attributed to indirect correlations with other
variables, such as relative humidity (Stuiver and
Braziunas 1987), precipitation (Anderson et al.
1998) or potential evapotranspiration (Brooks et al.
1998).
Differences in ∆13C due to the slope aspect
were generally low and dependent on the study
site. This suggests that the aspect effect on plant
water status was not only exerted through the
influence of an increased irradiance on
evapotranspiration. Another variable that should be
considered is wind regimes, which markedly
influences leaf temperature and evapotranspiration
rates (Jones 1992). This could be the case, for
example, of P. halepensis in region 1,
characterised by strong cold-dry winds
(tramuntana) coming from the North which could
increase water demand on North-faced slopes.
Nevertheless, we can not discard the effect of
additional intra-site heterogeneity due to soil
substrate, tree age or tree density (the latter, for
example, was often higher in North-facing slopes).
It should be noted that, when comparing
samples from different geographic origins,
differences in ∆13C might be partly due to adaptive
(i.e. genetic) variation in WUEi among populations
(Schulze et al. 1998; Anderson et al. 1996). This
can bring about an additional noise to the climatic
signal present in ∆13C, which can be either
enhanced or decreased (Schulze et al. 1998). In a
provenance trial of P. halepensis, Tognetti et al.
(1997) found lower stomatal reduction in seedlings
of xeric origins. If this was the case in our study,
genetic variation would have reduced the
variability in ∆13C among provenance regions. We
are unaware of similar studies for Q. ilex, but
genetic variability in water relations can be
considerable, even within populations (Leiva and
Fernández-Alés 1998). Nevertheless, environment
is by far the main factor responsive for ∆13C
variation in C3 plants. Indeed, the extreme regional
∆13C values found in our study matched well the
outermost values of precipitation and P/E within
each species (see Table 1.1, Fig. 1.2). In addition,
when we introduced P/E in a covariance model for
∆13C, this term explained about 45% and 67% of
the variability due to provenance regions for Q.
ilex and P. halepensis, respectively.
58
 Chapter 1. ∆13C and tree-ring width reflect different drought responses in Quercus ilex and Pinus halepensis
1.4.2. Can we track differences in drought
response through ∆13C?
Although both species showed a close
relationship between water availability and ∆13C,
the regression slopes of ∆13C on either
precipitation or P/E (on a log basis) were about
two-fold greater for P. halepensis than for Q. ilex.
Several works have reported faster stomatal
closure under drought for P. halepensis than for
Q. ilex. Whereas P. halepensis exhibits a rather
high threshold of predawn water potential for
stomatal closure (from -0.8 to -1.5 MPa; Borghetti
et al. 1998), it is considerably lower for Q. ilex
(from -3.0 MPa to -5.0 MPa; Damesin et al. 1998).
This translates into lower ∆13C sensitivity to water
availability in Q. ilex than in P. halepensis.
Moreover, Q. ilex is a deep-rooted tree and thus
can rely on groundwater during drought periods.
Correlations between ∆13C and monthly
precipitation for Q. ilex were about two-fold higher
in winter than in summer (see Fig. 1.5), suggesting
that this species relies mostly on groundwater
during the drier months. In contrast, the
relationship between ∆13C and precipitation was
nearly constant throughout the year in P.
halepensis, in concordance with the higher
dependence on rainfall of Mediterranean conifers
(Valentini et al. 1992; Nicault et al. 2001).
1.4.3. Radial growth response to
water availability and climate
Since the same range of radial growth could be
found for Q. ilex over the whole range of
precipitation, we can conclude that long-term
radial growth in populations from drier sites was
not water-limited. Plants growing under dry
environments may show several adaptations, either
phenological or physiological, allowing them to
keep growth rates similar as those shown in wetter
sites. For example, Q. ilex shows greater allocation
from leaves to xylem in sites with decreased water
availability (Villar-Salvador et al. 1997). In this
case, overall biomass could increase with water
availability, without significant changes in radial
growth rates. In addition, Q. ilex can rely on
groundwater for growth, even under very dry
climates (Valentini et al. 1992). Remarkably, the
only significant (negative) relationship between
radial growth in Q. ilex and precipitation was
found for September. In fact, greater precipitation
during early-autumn is often related with an
increased
seasonality
in
Mediterranean
environments. Q. ilex would display higher radial
growths in climates with lower seasonality,
regardless of the total amounts of precipitation.
This suggests that Q. ilex still preserves some
physiological features from its arcto-boreal origin
which are “out of phase” with the requirements of
a summer-dry habitat (Liphschitz and Lev-Yadun
1986).
In contrast, radial growth in P. halepensis was
highly dependent on water availability, as it was
correlated with both precipitation and ∆13C.
Moreover, a covariance analysis revealed that
precipitation described about 65% of the variability
in radial growth due to provenance regions.
Previous work found a strong intra-site relationship
between radial growth and precipitation in P.
halepensis (Serre 1976). Our results show that this
relationship also holds for inter-site variability. As
compared to winter, the lack of a relationship
between monthly precipitation and radial growth
during spring has also been observed in other
conifers (Lebourgeois 2000), since spring radial
growth is partly supported by previous-year
photosynthates (Lebourgeois 2000; Fallas 1970).
P. halepensis is considered a very plastic species
that shows two periods with reduced cambial
activity: one is defined by the summer drought and
the other by low winter temperatures (Liphschitz
and Lev-Yadun 1986). In our study, the positive
association between potential evapotranspiration
Published in Oecologia 137:512-518 (2003) 59
and radial growth during the coldest months
suggests that growth was also limited to some
extent by winter cold (cf. Fig. 1.5).
1.4.4. Concluding remarks
From our results, we can conclude that both Q.
ilex and P. halepensis respond to a decrease in
water availability by increasing their WUEi, as
inferred from wood ∆13C. However, P. halepensis
was more sensitive to water availability, showing a
steeper decrease in ∆13C with increasing aridity.
This is in agreement with the hypothesis that a
partially drought-tolerant species, such as Q. ilex,
would show lower increase in WUEi under drier
conditions than a drought-avoiding species (e.g. P.
halepensis). The fact that radial growth was related
with precipitation and ∆13C in P. halepensis but not
in Q. ilex further confirms that P. halepensis is
more dependent on precipitation in order to thrive
and survive.
Acknowledgements
This work was partly supported by the CICYT grant
BTE2001-3421-C02. P. Ferrio has a PhD. Fellowship
from Generalitat de Catalunya. We thank Instituto
Nacional de Meteorología and Confederación
Hidrográfica del Ebro for providing meteorological
data, and CU-INSTAAR/NOAA-CMDL for air δ13C
values. We also thank the gentle collaboration of the
Parc Natural de la Zona Volcànica de la Garrotxa, and
the forest services from Aragón and Catalunya.
60
 Chapter 1. ∆13C and tree-ring width reflect different drought responses in Quercus ilex and Pinus halepensis
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62
 Chapter 1. ∆13C and tree-ring width reflect different drought responses in Quercus ilex and Pinus halepensis
Chapter 2
“És el vent, per altra part, que treu dels pins de la costa el
rendiment més tangible; el gregal en treu un perfum sec i
crepitant; el garbí, més humit, gairebé femení, embriagant.”
Josep Pla. El meu país
63
Chapter 2
J.P. FERRIO1* • J. VOLTAS1
Carbon and oxygen isotope ratios in
wood constituents of Pinus halepensis as indicators
of precipitation, temperature and vapour pressure deficit
1
Departament de Producció Vegetal i Ciència Forestal, E.T.S.E.A - Universitat de Lleida,
Av. Rovira Roure 191, Lleida E-25198, Spain
*Corresponding author; e-mail: [email protected]
Abstract Carbon and oxygen isotope compositions (δ13C, δ18O) in tree-rings have been shown to bear
relevant climatic signals. However, little is known about the interrelation between both isotopes in
wood constituents, and only referred to species from relatively wet climates. We hypothesized that, in
a species adapted to temporary droughts (e.g. Pinus halepensis Mill.), the signal derived from δ18O in
precipitation would be hidden by the strong variability in leaf transpirative enrichment. To test this
assumption, we compared the effect of precipitation, temperature and vapour pressure deficit (VPD)
on δ18O and δ13C along 23 sites covering the ecological range for this species. We extracted the cores
from the south side of 4-6 adult, dominant trees per aspect (north/south) within each site. For each
aspect and site, fragments of the period 1975-1999 were pooled and milled to a fine powder. To
further test the postulated need for cellulose purification in the assessment of climatic information, we
studied these relationships in whole and extracted wood, holocellulose and lignin. In all wood
fractions, d13C was related to annual precipitation (r=-0.58** to -0.78***) and VPD (r=0.53** to
0.57**). In contrast, for δ18O only holocellulose showed consistent relationships with climatic data,
being strongly significant for VPD (r=0.66***). However, it was unrelated to modelled δ18O in
precipitation, confirming that transpirative enrichment (driven by VPD) dampened the source signal in
P. halepensis. The relationships between δ13C and δ18O were generally poor, regardless of the wood
constituent, suggesting that, although both variables were somewhat related to transpirative demand,
they were relatively independent. This was further confirmed by building stepwise models using both
isotopes to predict annual and seasonal precipitation (r2=0.34** to 0.68***), temperature (r2=0.15* to
0.37**) and VPD (r2=0.31** to 0.55***). We concluded that, even describing partially the same
climate variables, the information underlying the two isotopes can be regarded as complementary.
2.1. Introduction
Carbon and Carbon and oxygen isotope ratios
from tree-rings offer the possibility of highresolution records of past climate. Although both
isotopes have been related to similar environmental
variables, the processes leading to such
relationships differ substantially between them.
Carbon isotope composition (δ13C) of plants is
isotopically depleted in 13C respect the atmospheric
CO2, which is the carbon source for
photosynthesis. The instantaneous values of δ13C
in C3 plants are determined by the ratio of
intercellular to atmospheric partial pressure of CO2
(pi/pa), reflecting the balance between diffusion in
the intercellular space (mostly through stomata)
and carbon fixation by the carboxylating enzyme.
Published in Tellus Series B-Chemical and Physical Meteorology 57B:164-173 (2005) 65
Consequently, δ13C in dry matter constitutes an
integrated record of the plant water status during
growth (Farquhar et al. 1982). δ13C in wood is
therefore expected to integrate the effect of
different environmental variables on the water
status of trees. Indeed, δ13C has been related to
various climatic variables, such as humidity or
temperature (Anderson et al. 1998; Hemming et al.
1998), and several indicators of water availability
(Dupouey et al. 1993; Ferrio et al. 2003a;
Hemming et al. 1998; Saurer et al. 1997a).
The association between oxygen isotopes and
environmental variables, however, is less clear.
The oxygen isotope composition (δ18O) of plant
tissues reflects the variation in (1) δ18O in source
water, (2) evaporative enrichment of leaf water due
to transpiration, and (3) biochemical fractionation
during the synthesis of organic matter (Farquhar
and Lloyd 1993; Yakir 1992). Generally, the δ18O
of source water is strongly dependent on that of
rain water (δ18OR). δ18OR is mainly determined by
the temperature of droplet formation, being higher
with higher temperatures (Dansgaard, 1964). Other
factors, such as altitude or precipitation, also affect
δ18OR (Barbour et al. 2001). The level of
enrichment of leaf water above source water
(∆18Oe) has been modelled as follows (Dongmann
et al. 1974; Farquhar and Lloyd, 1993):
Eq. 2.1
∆18Oe =ε*+ε k +(∆18Ov -ε k ) × (ea /ei )
where ε* is the proportional depression of
vapour pressure by the heavier H218O, εk is the
diffusion fractionation through stomata and leaf
boundary layer, ea and ei stand for vapour pressure
in atmosphere and intercellular space, respectively,
and ∆18Ov is the oxygen isotope composition of
water vapour in the atmosphere (relative to source
water). According to this model, plants growing at
higher humidity (i.e. higher ea) are expected to
have lower ∆18Oe (for further details, see Farquhar
and Lloyd 1993). Within the same environmental
conditions, plants
with higher stomatal
conductance (i.e. lower leaf temperature, which
reduces ei) are also expected to show smaller
∆18Oe. Leaf water enrichment is passed on to the
organic molecules formed in the leaf by exchange
of oxygen atoms between carbonyl groups and
water (Sternberg et al. 1986). However, in the case
of cellulose, which is the main component of
wood, about 50% of the enrichment signal (derived
from sucrose) is further exchangeable with xylem
water. Such exchange is determinant for the
observed relationship between δ18O of tree-ring
cellulose and δ18OR, as it enhances the sourcewater signal, softening the effect of leaf-level
enrichment (Anderson et al. 1998; Barbour et al.
2001; Saurer et al. 1997b).
Traditionally, tree-ring analyses have been
performed on purified cellulose. However, recent
works have shown strong relationships between the
isotopic composition (δ13C or δ18O) of cellulose
and other wood fractions, suggesting that this timeconsuming process might be obviated (Barbour et
al. 2001; Borella et al. 1998). On the other hand,
although the association between either δ13C or
δ18O and environmental variables has been widely
studied, the information available about the
interrelation between both isotopes in wood
cellulose is still scarce (Anderson et al. 1998;
Saurer et al. 1997a), and we are not aware of such
studies for other wood fractions. Moreover, the
results from these works, performed in temperate
areas, are not necessarily extrapolable to
Mediterranean species, as they are affected by
different environmental constraints and may
respond in a different way to the same climatic
variables. This might be the case of the species
chosen in this study, Aleppo pine (Pinus
halepensis Mill), which is highly sensitive to
changes in water availability as an adaptive
strategy to the great inter-annual and seasonal
variability of Mediterranean climates. Thus, it is
66
 Chapter 2. Carbon and oxygen isotope ratios in wood constituents of Pinus halepensis as indicators of climate
able to reduce drastically growth and water
expense during drought by stomatal closure,
recovering rapidly when water becomes available
(Borghetti et al. 1998; Ferrio et al. 2003a). We
hypothesized that, in a species with such
responsive stomata, the potential δ18OR signal in
wood tissues would be hidden by the strong
variability
in
transpirative
enrichment.
Consequently, δ18O would be more closely related
to tree water status (and thus to δ13C variation)
than to the thermal signal retained in δ18OR. To
assess this hypothesis, we compared the effect of
climatic variables on wood δ13C and δ18O over the
range of environmental conditions where this
species can be found. On the other hand, to further
test the postulated need for cellulose purification,
we aimed to characterise possible changes in the
climatic signal present in both δ13C and δ18O
during the extraction process. The main novelty of
our approach lies in the simultaneous examination
of both isotopes, as this may help to understand the
relationships among the isotope signatures present
in different wood components and their
implications for environmental studies.
2.2. Materials and Methods
Fig. 2.1 Geographical distribution of the reference sites (Table
2.1) used to test the accuracy of climatic models and the
sampling sites for Pinus halepensis wood cores (Table 2.2).
Main bioclimatic regions in the studied area are also indicated,
according to the UNESCO humidity index (Total
precipitation/Pemman evapotranspirative demand). Redrawn
from Spanish Ministry of the Environment (2000).
2.2.1. Plant material and sample preparation
We sampled wood cores (5mm diameter) of
Pinus halepensis Mill. from 23 sites in the East
Iberian Peninsula (Western Mediterranean Basin),
selected to be representative of the natural
ecological range for this species (see Fig. 2.1). We
extracted the cores from the south side of 4-6 adult,
dominant trees per aspect (north/south) within each
site. Samples were oven-dried at 60ºC for 48h
before being polished to allow tree-ring dating. We
selected a fragment of the cores, corresponding to
the period 1975-1999. Fragments from the same
site and aspect (23 sites x 2 aspects = 46) were
then pooled and milled to a fine powder.
2.2.2. Carbon and oxygen isotope analysis
For the removal of extractives and lignin, we
adapted the method of Leavitt and Danzer (1993)
for holocellulose purification. We divided the
samples in three subsets: the first set of samples
was analysed for isotope content without previous
treatment (whole wood); the second one, after
extractives (resins, soluble carbohydrates and
other mobile compounds) were removed (extracted
wood); and the third one, after the removal of
lignin (holocellulose). We weighted 90-110mg dry
mass (oven at 60ºC, 24h) within individual
Published in Tellus Series B-Chemical and Physical Meteorology 57B:164-173 (2005) 67
pouches made of glass-fiber filter paper (no. 30
Schleicher & Schuell, Dassel). The pouches were
cinched up with 4-in nylon cable ties with a
number inscribed for identification, and reinforced
with waxed flossing tape (dental). We distributed
the pouches among 4 soxhlet extractors (250ml)
filled with a 2:1 toluene/ethanol mixture and run
overnight (16h) at 70ºC. After shutdown and
cooling, samples were air-dried for 1-2h before
returning them to the soxhlet, filled with 100%
ethanol (16h at 70ºC). At this point, samples
became free of resins and other solvent-extractable
substances. After cooling and air-drying (1-2h), we
put the samples in the soxhlet and extracted them
with deionised water for 6h at 100ºC. Then we
took away half of the pouches (“extracted wood”).
The rest of pouches (still wet) were transferred to a
2000ml Erlenmeyer filled with 1000ml deionised
water, 5g sodium chlorite (NaClO2, technical
grade) and 1.5ml glacial acetic, keeping the flask at
70ºC for 12h. After 4 additions of sodium chlorite
and acetic acid, in 12h intervals, we rinsed the
samples until the conductivity of the supernatant
was less than 0.5 µS cm-1. All samples were ovendried (60ºC, 48h) and weighted for the
quantification of holocellulose. δ13C and δ18O in
whole wood, extracted wood and holocellulose
were determined by mass spectrometry at Isotope
Services, Inc., (Los Alamos, NM, USA) and
Serveis Científico-Tècnics (Universitat de
Barcelona,
Spain),
respectively.
Isotopic
composition
of
lignin
was
estimated
gravimetrically (see e.g. Barbour et al. 2001;
Borella et al. 1998; Gray and Thompson 1977)
from the above measurements and the dry-weight
ratio between holocellulose and extracted wood
(MC/MeW):
Eq. 2.2
δL = (δeW - δC (M C /M eW ))/(1-(M C /MeW ))
where δL, δeW and δC stand for isotopic
composition (δ13C or δ18O) of lignin, extracted
wood and holocellulose, respectively.
2.2.3. Meteorological data
and δ18O in precipitation
Meteorological data for the period 1975-1999
were supplied by the Instituto Nacional de
Meteorología and the Confederación Hidrográfica
del Ebro. We took monthly values of precipitation
(in mm) and temperature (in ºC). Annual means of
δ18O in rain water (δ18OR) and vapour pressure
have been successfully modelled on a global scale
by Barbour et al. (2001) from annual precipitation
(Pan), mean annual temperature (Tan) and altitude
(Z, in m). However, annual means are probably
imperfect indicators of the great differences in
climate seasonality found among Mediterranean
sites. Thus, we built alternative models to predict
vapour pressure in a monthly basis. We gathered
from the Global Network of Isotopes in
Precipitation (GNIP) database (IAEA/WMO,
2001) monthly values of precipitation (Pm),
temperature (Tm), vapour pressure (VP) and δ18OR
from 52 sites across the Mediterranean Region. We
calibrated the model using monthly means for each
site, and taking only the sites with data available
for the whole year. The resulting model for VP is
as follows (r2=0.94***, n=312, 26 sites):
Eq. 2.3
ln(VP)=6.34+0.047Tm +0.93(Pm /1000)-0.22(Z /1000)
We used the data from the sites not included in
calibration to validate this model, obtaining a
strong correlation between predicted and measured
values (r=0.93***, n=102). Moreover, calculating
annual means for 10 sites located around the
studied area (Fig. 2.1) we obtained better results
using Eq. 2.3 than with the global model from
Barbour et al. (2001; see Table 2.1). Taking
monthly means of day-time temperature (Tday=1/3
average minimum temperature + 2/3 average
maximum temperature), we obtained day-time
saturation vapour pressure (VPsat) on a monthly
basis (Jones 1992):
68
 Chapter 2. Carbon and oxygen isotope ratios in wood constituents of Pinus halepensis as indicators of climate
Table 2.1 Site description and oxygen isotope composition in precipitation (δ18OR; rainfall-weighted annual mean) for 10
reference sites used to assess the accuracy of vapour pressure (VPan, annual mean of vapour pressure) and δ18OR models
described in Eqs. 2.3 and 2.5. Lat., latitude; Long., longitude; Z, Altitude; Pan, mean annual precipitation; Tan, mean annual
temperature; VPDan, annual mean of day-time vapour pressure deficit; Obs., observed values; Global, predicted values using the
global, yearly models developed by Barbour et al. (2001); MED, predicted values using our models for the Mediterranean
Region, built on a monthly basis. Correlation coefficients between predicted and observed values are also indicated.
Reference
Site
Lat.
Long.
A. Almería
36º51' 02º23'E
B. Barcelona
41º23' 02º07'E
C. Ciudad Real 38º59' 03º55'W
D. Girona
41º54' 02º45'E
E. Madrid
40º25' 03º41'W
F. Murcia
38º00' 01º10'W
G. Palma
39º33' 02º37'E
H. Tortosa
40º49' 00º29'E
I. València
39º28' 00º23'W
J. Zaragoza
41º39' 01º01'W
r (pred vs obs)
sig.
*P<0.05, **P<0.01, ***P<0.001
Z
(m)
21
90
682
129
655
62
3
48
13
263
VPan (Pa)
Obs. Global MED
VPDan (Pa)
Obs. MED
δ18OR (‰)
Obs. Global MED
1503
1347
1061
1310
956
1253
1600
1345
1504
1153
945
676
1163
650
1009
1239
779
1035
902
1006
-3.7
-5.0
-6.9
-4.7
-6.4
-4.6
-4.9
-4.9
-5.1
-5.5
Pan Tan
(mm) (ºC)
182
593
383
562
433
287
311
496
477
301
19.3
16.0
15.9
15.0
14.5
18.9
18.6
18.1
18.9
15.8
Eq. 2.4


Tday
VPsat =613.75 × exp 17.502

240.97+Tday 

Finally, we estimated monthly means of daytime vapour pressure deficit (VPD) by subtracting
VP from VPsat.
In the same way, we obtained a monthly model
for δ18OR (r2=0.52***, n=360, 30 sites):
Eq. 2.5
δ18OR =0.42Tm -0.007Tm2 -26.8(Pm /1000)
+112(Pm /1000) 2 -0.046 Z-8.26
On a monthly basis, the estimates of δ18OR were
less accurate than for VP (r=74***, n=128 for
validation sites). Nevertheless, when annual means
were calculated, the results obtained with the
global model (Barbour et al. 2001) were still
improved, as shown in Table 2.1.
1387
1196
1195
1142
1114
1363
1346
1320
1366
1187
0.75
**
1451
1277
1125
1207
1051
1439
1427
1417
1464
1207
0.85
***
825
757
891
754
915
988
876
963
878
969
0.83
***
-5.0
-5.7
-6.7
-6.2
-7.1
-5.1
-4.9
-5.0
-4.6
-6.4
0.75
**
-4.3
-5.1
-6.4
-5.5
-6.4
-4.6
-4.4
-4.6
-4.3
-5.2
0.85
***
2.2.4. Statistical analysis
The data were subjected to analysis of variance
(ANOVA) to assess the effect of chemical
extraction, site and aspect on stable isotope
compositions. Principal components analyses
(PCA) and simple correlations were used to assess
relationships among variables. We built stepwise
linear regressions to predict site variables from
oxygen and/or carbon isotopes in different wood
fractions, but the selection of variables for the
model was further restricted in such a way that
only the best-fitting wood fraction per isotope
(carbon or oxygen) was allowed to enter the
model. This was done to quantify the existence of
complementary responses to climate variables
between carbon and oxygen stable isotopes. Unless
otherwise stated, differences were considered
statistically significant when P<0.05.
Published in Tellus Series B-Chemical and Physical Meteorology 57B:164-173 (2005) 69
2.3. Results and discussion
2.3.1. δ13C and δ18O values in wood
and wood fractions
As expected, we found significant changes in
δ13C and δ18O during removal of extractives and
lignin from wood to obtain holocellulose (Table
2.2). On the contrary, we did not find any
significant effect of sampling aspect on either δ18O
or δ13C, so we decided to work with site means
(North and South-facing slopes combined)
throughout the study. δ13C of whole wood
(δ13CW) and extracted wood (δ13CeW) differed
significantly, and both had clearly lower values
than that of holocellulose (δ13CC), with a mean
difference of 1.2‰ and 1.0‰, respectively. Unlike
for δ13C, there were no differences between δ18O of
whole wood (δ18OW) and extracted wood (δ18OeW),
but δ18O of holocellulose (δ18OC) was about 4‰
higher. Similar results have been reported for δ13C
(Borella et al. 1998; Leavitt and Danzer, 1993) and
δ18O (Barbour et al. 2001; Gray and Thompson,
1977) variations in several conifers. The effect of
holocellulose purification on δ13C was constant
across sites, whereas there was a significant
interaction with site for δ18O, probably caused by
Table 2.2 Site description and mean values (North and South-facing slopes combined) for δ13C and δ18O (‰) in whole wood
(δ13CW, δ18OW), extracted wood (δ13CeW, δ18OeW), holocellulose (δ13CC, δ18OC) and lignin (δ13CL, δ18OL). Lat., latitude; Long.,
longitude; Z, Altitude; Pan, mean annual precipitation; Tan, mean annual temperature; VPDan, annual mean of day-time vapour
pressure deficit; δ18OR, estimated δ18O of precipitation (rainfall-weighted annual mean).
Site
Lat.
1 41º56'
2 41º58'
3 42º01'
4 41º09'
5 41º01'
6 41º12'
7 41º20'
8 41º55'
9 41º47'
10 42º19'
11 42º09'
12 41º54'
13 41º10'
14 40º53'
15 41º03'
16 41º23'
17 41º41'
18 41º50'
19 37º26'
20 42º28'
21 40º41'
22 41º36'
23 42º05'
Mean
S.D.
Long.
03º15'E
02º49'E
03º00'E
01º17'E
00º36'E
01º39'E
00º30'E
01º01'E
02º26'E
00º45'W
00º15'E
00º26'E
00º23'E
00º12'E
00º06'E
00º17'E
00º12'W
00º32'W
02º20'W
01º15'W
00º34'E
01º43'E
01º20'E
Z
(m)
185
44
190
148
180
106
80
525
208
520
625
518
363
630
359
125
356
380
900
360
201
230
480
Pan
(mm)
693
732
809
478
490
511
395
551
753
519
551
423
427
563
361
385
362
407
337
561
562
417
610
Tan VPDan δ18OR
(ºC)
(Pa)
15.2
805
-5.7
15.4
778
-5.3
14.1
723
-6.0
15.8
812
-5.0
15.2
807
-5.4
15.9
849
-5.0
14.8
826
-5.2
12.7
791
-6.4
15.1
807
-5.8
13.7
841
-6.3
12.9
814
-6.5
12.5
788
-6.3
14.7
898
-5.9
13.2
788
-6.4
15.9
997
-5.3
14.5
827
-5.3
14.9
897
-5.5
13.5
801
-5.8
17.8 1220
-5.6
13.2
743
-6.2
16.7
917
-5.2
14.0
695
-5.4
13.2
773
-6.2
517 14.6
133 1.4
835
106
-5.7
0.5
δ13CW δ13CeW δ13CC
δ13CL δ18OW δ18OeW δ18OC δ18OL
-24.9
-24.2
-24.7
-24.0
-23.8
-23.8
-22.7
-24.4
-23.7
-23.6
-24.6
-24.0
-23.4
-24.3
-22.8
-23.2
-23.3
-23.2
-22.7
-25.0
-24.3
-23.6
-25.0
-23.9
0.7
-26.2
-25.3
-25.4
-25.2
-25.1
-25.2
-24.7
-25.9
-25.1
-25.1
-25.0
-25.2
-24.9
-25.1
-24.8
-24.3
-24.5
-24.0
-24.2
-26.5
-25.2
-25.7
-25.8
-25.2
0.6
-24.7
-24.0
-24.4
-23.7
-23.6
-23.7
-22.7
-24.2
-23.7
-23.5
-24.2
-23.8
-23.1
-24.1
-22.7
-22.8
-22.9
-22.7
-22.6
-24.9
-23.8
-23.4
-24.4
-23.6
0.7
-23.9
-23.2
-23.6
-22.8
-22.7
-22.7
-21.6
-23.1
-22.7
-22.3
-23.3
-22.7
-22.1
-23.1
-21.7
-21.9
-22.3
-21.6
-21.5
-23.7
-23.0
-22.1
-23.4
-22.7
0.7
26.7
27.2
28.1
27.9
28.2
27.1
28.4
28.2
27.0
28.0
28.3
27.5
28.0
27.9
28.1
28.8
27.9
27.7
28.0
27.2
27.6
27.2
27.1
27.7
0.5
26.4
27.9
27.7
28.9
28.2
28.2
29.3
29.4
27.9
28.0
28.0
27.1
27.6
27.5
28.8
29.7
28.4
27.5
28.2
26.4
28.7
27.0
26.7
28.0
0.9
30.1
30.6
30.3
31.4
31.3
29.7
32.4
31.3
29.5
31.2
31.9
30.9
32.2
31.5
31.7
32.1
31.2
30.7
34.6
31.5
35.0
30.7
29.6
31.4
1.4
70
 Chapter 2. Carbon and oxygen isotope ratios in wood constituents of Pinus halepensis as indicators of climate
18.6
23.2
24.2
24.1
22.8
25.9
23.5
26.6
25.7
23.5
24.2
21.4
20.7
23.3
22.8
26.1
23.0
23.7
18.9
19.6
17.8
20.7
22.7
22.7
2.4
Table 2.3 Correlation coefficients among site means of δ13C and δ18O values for each of the wood fractions studied: whole wood
(δ13CW, δ18OW), extracted wood (δ13CeW, δ18OeW), holocellulose (δ13CC, δ18OC) and lignin (δ13CL, δ18OL).
δ13CW
δ13CeW
0.12
0.10
δ18OL
0.35
0.38
δ18OC
0.54**
0.54**
δ18OeW
0.47*
0.49*
δ18OW
0.80*** 0.87***
δ13CL
0.97*** 0.97***
δ13CC
0.98***
δ13CeW
*P<0.05, **P<0.01, ***P<0.001
δ13CC
0.08
0.36
0.46*
0.44*
0.79***
δ13CL
0.21
0.38
0.52**
0.57**
the sensitivity of δ18OC to climate variables, that
was not reflected in δ18OW or δ18OeW, as we will
discuss in point 2.3.2. On the other hand, our
results suggest that, in contrast to δ13C, the sources
of variability for δ18O differed strongly between
the main fractions of wood (lignin and
holocellulose). δ13C in lignin (δ13CL) was
strongly, positively related to δ13CC (Table 2.3)
despite being 2.5‰ lower in average. Conversely,
δ18OC and lignin δ18O (δ18OL) differed considerably
(8.7‰) and, unlike δ13C values, were negatively
correlated. This observation further evidences that,
for carbon and oxygen, the origin of differences in
isotope composition between lignin and
holocellulose is totally independent. Changes in
δ13C may be due to chemical fractionation during
lignin biosynthesis (Benner et al. 1987) and/or
seasonal differences in the amount of carbon
directed to the synthesis of cellulose or lignin
precursors (Förster et al. 2000). However, the main
source of variation in δ13C for C3 plants is shared
by all wood components, and it appears to be
fractionation during carbon fixation. This explains
the observed correspondence between δ13CC and
either δ13CL, δ13CeW or δ13CW, in agreement with
previous works (Borella et al. 1998; Mazany et al.
1980). On the contrary, the negative correlation
between δ18OC and δ18OL is more controversial,
and both negative (Gray and Thompson 1977) and
positive (Barbour et al. 2001) relationships have
been reported. In both studies, δ18OL was estimated
δ18OW
0.34
0.49*
0.73***
δ18OeW
δ18OC
0.51*
0.41*
-0.48*
gravimetrically, as was done in our study, and thus
we could discard the errors associated with this
method as the cause of such differences. Moreover,
such errors would have affected in the same way
δ13CL and δ18OL, and they displayed very different
behaviours. The most The most probable origin for
these contrasting results lies in the exceptional
climatic range used by Barbour et al. (2001),
including over 19‰ of variation in δ18OR (1.5‰ in
our work). Although the sources of variation for
δ18OC are well described, little is known about
δ18OL (Barbour et al. 2001). Assuming some
exchange of oxygen atoms between xylem water
(=δ18OR) and lignin (as in cellulose), we would
expect a positive relationship between δ18OC and
δ18OL over a wide range of δ18OR, but not if δ18OC
and δ18OL variability can not be explained by
δ18OR, as seems to be our case (see point 2.3.2).
2.3.2. Effect of environmental variables
on δ13C and δ18O
A PCA weights plot (Fig. 2.2) provided an
initial insight into the association trends for δ13C
and δ18O along with the environmental variables
Pan, Tan and VPDan. δ13C of the different wood
fractions clustered together and opposed to Pan
over the axis of the first component. This agrees
with the good correspondence among δ13C of
different wood fractions (Table 2.3) and the
expected negative relationship between δ13C and
plant water status. In contrast, the performance of
Published in Tellus Series B-Chemical and Physical Meteorology 57B:164-173 (2005) 71
δ18O varied substantially among fractions.
According to their relative positions in the biplot,
δ18OC was related to changes in both VPDan and
Tan, whereas δ18O of other wood constituents was
not clearly related to any environmental variable,
being relatively independent of δ18OC variation.
Surprisingly, estimated δ18OR was only
significantly related to δ18OeW, but not to δ18OC
(Table 2.4). The same pattern was found when
looking at correlations with estimated seasonal
means of δ18OR (data not shown). However, the
highest correlation with δ18OeW (r=0.49*, N=23)
was found in summer, when modelled δ18OR
showed the poorest correlation with measured
δ18OR (r=0.49**, ranging from 0.71*** to 0.84***
for the other seasons, N=30). Such relationship
Principal Component 2
0.8
18
δ OL
0.6
δ OW
18
δ OeW
18
0.4
0.2
0.0
13
δ CL
13
δ CC,eW,W
Pan
-0.2
VPDan
Tan δ18O
C
-0.4
-0.6
-0.8
-0.4 -0.2
0.0
0.2
0.4
0.6
0.8
Principal Component 1
Fig. 2.2 Component weights plot of the principal components
analysis including the three climatic variables studied and site
means for δ13C and δ18O in whole wood (δ13CW, δ18OW),
extracted wood ( δ13CeW, δ18OeW), holocellulose (δ13CC,
δ18OC) and lignin (δ13CL, δ18OL). Pan, mean annual
precipitation; Tan, mean annual temperature; VPDan, annual
mean of day-time vapour pressure deficit. The two
components plotted explained 54% and 19% of total variance,
respectively.
might be an artefact, and could be explained by the
strong negative relationship found between lignin
to cellulose ratio (L/C) and estimated δ18OR (Table
2.4). As lignin is isotopically lighter than cellulose,
a depletion in lignin content is expected to increase
both δ13CeW and δ18OeW. Indeed, correlation
coefficients between δ18OR and either δ13CeW or
δ13CW were even greater than with δ18OeW. At this
point, it should be noted that the relationship
between L/C and δ18OR has no physiological
meaning, being just the consequence of a common
influence of temperature and altitude on both
variables (Barbour et al. 2001; Dansgaard 1964;
Kollmann 1955).
The lack of a consistent relationship between
d18O of wood constituents and δ18OR confirms our
hypothesis that, for a species with tight stomatal
regulation such as P. halepensis, a relatively small
range of δ18OR (1.5‰) is hidden by the stronger
variability in ∆18Oe (see Eq. 1). The association of
δ18OC with VPDan (the driving force for
transpiration) was stronger than with Tan (Table 4),
further suggesting that δ18OC variations were more
closely related to transpirative demand than to the
temperature signal stored in δ18OR. In contrast,
other authors found, over a similar range of δ18OR,
good correlations between δ18OR and δ18OC in treerings of Fagus sylvatica (Saurer et al. 1997b) and
Picea abies (Anderson et al. 1998). However, both
species are less adapted to water stress than P.
halepensis, and thus are expected to show a looser
stomatal control. Moreover, Saurer et al. (1997b)
obtained a poorer relationship with δ18OR at the
driest site, in agreement with our assumption that
the source water signal in δ18OC is easily dampened
when water is limiting and VPD increases.
δ13C in the different wood fractions provided
similarly strong relationships with climatic
variables (Table 2.4). Pan showed strong negative
correlations with δ13CW, δ13CeW and δ13CC, being
somewhat weaker for δ13CL. On the other hand,
δ13C showed positive trends with Tan and VPDan.
For Tan, this relationship was only significant for
72
 Chapter 2. Carbon and oxygen isotope ratios in wood constituents of Pinus halepensis as indicators of climate
Table 2.4 Correlation coefficients between site parameters and either δ13C or δ18O in whole wood (δ13CW, δ18OW), extracted wood
(δ13CeW, δ18OeW), holocellulose (δ13CC, δ18OC) and lignin (δ13CL, δ18OL), as well as with dry-weight lignin to cellulose ratio (L/C).
Pan, mean annual precipitation; Tan, mean annual temperature; VPDan, mean annual vapour pressure deficit; δ18OR, estimated δ18O
of precipitation (rainfall-weighted annual mean).
Tan
0.43*
0.42*
0.33
0.32
-0.04
0.32
0.46*
-0.30
-0.54**
VPDan
0.56**
0.57**
0.53**
0.55**
0.25
0.32
0.66***
-0.31
-0.16
0.8
0.6
0.4
0.2
0.0
-0.2
-0.4
-0.6
-0.8
a) Whole wood (W)
0.8
0.6
0.4
0.2
0.0
-0.2
-0.4
-0.6
-0.8
b) Extracted wood (eW)
have enhanced the depletion in δ13C associated
with lower temperatures (mostly due to lower
evaporative demand).
Correlation coefficients between seasonal
means of climate variables and δ13C were also
consistent among wood components, despite being
weaker for δ13CL (Fig. 2.3). Hence, the relationship
of δ13C with precipitation and VPD was significant
c) Holocellulose (C)
δ C
18
δ O
d) Lignin (L)
Pspring
Psummer
Pautumn
Pwinter
Tspring
Tsummer
Tautumn
Twinter
VPDspring
VPDsummer
VPDautumn
VPDwinter
Pspring
Psummer
Pautumn
Pwinter
Tspring
Tsummer
Tautumn
Twinter
VPDspring
VPDsummer
VPDautumn
VPDwinter
13
0.8
0.6
0.4
0.2
0.0
-0.2
-0.4
-0.6
-0.8
0.8
0.6
0.4
0.2
0.0
-0.2
-0.4
-0.6
-0.8
Pspr
Psum
Paut
Pwin
Tspr
Tsum
Taut
Twin
VPDspr
VPDsum
VPDaut
VPDwin
Pspr
Psum
Paut
Pwin
Tspr
Tsum
Taut
Twin
VPDspr
VPDsum
VPDaut
VPDwin
Correlation coefficients (r)
δ13CW and δ13CeW, whereas for VPDan we found
significant correlations for all wood constituents.
As for δ18OR, the greater sensitivity of whole and
extracted wood to Tan may be due to the negative
relationship observed between L/C and Tan (Table
2.4). As lignin is more depleted in 13C than the
remaining wood, the greater lignin content found
in colder sites (see e.g. Kollmann 1955) would
δ18OR
0.45*
0.45*
0.36
0.25
-0.03
0.42*
0.17
-0.05
-0.74***
Correlation coefficients (r)
Altitude
Pan
0.02
-0.72***
δ CW
0.04
-0.75***
δ13CeW
0.11
-0.78***
δ13CC
0.16
-0.58**
δ13CL
0.17
-0.46*
δ18OW
-0.18
-0.35
δ18OeW
0.31
-0.46*
δ18OC
-0.19
0.11
δ18OL
L/C
-0.56**
0.20
*P<0.05, **P<0.01, ***P<0.001
13
Fig. 2.3 Correlation coefficients between seasonal means of climatic variables and site means for δ13C and δ18O of the different
wood fractions. Dotted lines indicate the threshold value for significant correlations (P<0.05). P, T and VPD stand for
precipitation, temperature and vapour pressure deficit, respectively; each seasonal mean is indicated by the following subindex:
Xspr (spring), Xsum (summer), Xaut (autumn) and Xwin (winter).
Published in Tellus Series B-Chemical and Physical Meteorology 57B:164-173 (2005) 73
throughout the seasons, albeit being a bit lower in
winter. For temperature, summer was the most
influencing period on δ13C. The higher sensitivity
of δ13C to summer temperature and VPD is a direct
consequence
of
Mediterranean
climates,
characterized by a dry-hot summer, when VPD
strongly determines the water status of plants, and
thus δ13C. The generally good sensitivity of δ13C to
precipitation throughout the year may be explained
by the particular physiology of the species under
study. Unlike in deciduous trees (see e.g. Helle and
Schleser 2004), early wood in Pinus species is
almost
entirely
produced
from
current
photosynthate (Dickmann and Kozlowski 1970;
Glerum 1980) and both early and late wood isotope
composition has proven to be related to the
environmental conditions in which they were laid
down (Barbour et al. 2002; Leavitt and Long
1991). Moreover, P. halepensis has an indefinite
growth, which often span over the whole year,
even though it is slowed during the drier weeks of
summer, as well as in cold winters (Borghetti et al.
1998; Lev-Yadun 2000; Liphschitz and Lev-Yadun
1986; Nicault et al. 2001). Consequently, the
isotope composition of tree-rings in this species
has the potential of being representative of the
environmental conditions throughout the year.
However, differences among sites in the seasonal
growth pattern might have added an uncontrolled
source of variation to the isotopic values of wood
material. In order to check for this effect, we
calculated the ratio between early and late wood
widths in 11 sites (3, 5, 7, 11, 14, 18, 19, 20, 21, 22
and 23; Table 2) where scanned images of the
cores were available. Despite having a
considerable range of variation for this ratio across
sites (1.2-3.5), it was not significantly related to
either δ18O or δ13C variation. This suggests that
growth variability among sites had little influence
on the bulk isotopic values obtained.
Unlike for δ13C, holocellulose purification was
required to obtain consistent relationships between
climatic variables and δ18O. Although δ18OW and
δ18OeW were somewhat related to precipitation and
temperature, respectively, such relationships were
poor and inconsistent (Table 2.4, Fig. 2.3). In
contrast, δ18OC showed significant relationships
with both annual and seasonal means of VPD
(r=0.58-0.69), as well as with summer precipitation
(r=-0.63). As expected from the lack of
correspondence between δ18OC and δ18OL, we did
not find any significant correlation between
climate variables and δ18OL. As already suggested
by Barbour et al. (2001), only along extremely
wide ranges of δ18OR (unrealistic for most tree-ring
studies) it is likely to obtain similar information
from holocellulose and wood or lignin. The
suggested sources of variability for δ18OL are the
isotopic composition of atmospheric O2 (used in
the synthesis of lignin precursors) and the potential
exchange with either leaf or xylem water (Barbour
et al. 2001). As δ18OL and δ18OC were negatively
correlated, the proportion of oxygen exchanged
with water in lignin should have been relatively
small; otherwise, they would share the same waterderived δ18O signal. Thus, the variability in δ18OL
could be related to variations in O2 isotopic
signature. The bulk isotopic value of atmospheric
O2 is relatively stable around the world (Barbour et
al. 2001). However, and despite the high
concentration of O2 in the atmosphere, in closed
canopies or, at least, within the leaf boundary
layer, air O2 isotopic composition is affected by the
balance between different respiratory processes
with contrasting fractionations for O2 (Angert and
Luz 2001; Farquhar and Lloyd 1993; Nagel et al.
2001; Ribas-Carbo et al. 2000). Probably, such
balance is not directly related to the environmental
variables here monitored, which would agree with
the apparent lack of environmental effects on δ18OL
variations (Nagel et al. 2001). Although the
discussion of the mechanisms of CO2 fractionation
is far from the aim of this work, further studies to
determine the origin of δ18O signature in lignin are
encouraged, as they might offer a new, integrated
source of physiological information (e.g. if the
isotopic signal from canopy or leaf O2 is somehow
preserved in lignin).
74
 Chapter 2. Carbon and oxygen isotope ratios in wood constituents of Pinus halepensis as indicators of climate
2.3.3. Are δ13C and δ18O complementary?
As a further test of the applicability of
combining δ13C and δ18O isotopes in
environmental studies, we built stepwise regression
models to estimate climate variables from oxygen
and/or carbon isotopes (Table 2.5). For
precipitation, δ13CC was the most useful variable,
although closely followed by δ13CW. δ18OC was
only considered for summer precipitation, where
the best model included δ13CW and δ18OC.
Conversely, temperature was poorly predicted. For
annual means, only δ18OC showed some (weak)
relationship with temperature. The best fit among
temperature models was found in summer,
combining δ13CW and δ18OC. On the other hand,
VPD was generally related to δ13CW and δ18OC.
Indeed, the best predictions were found combining
both variables, except in winter, where δ18OC was
taken alone. In general, δ13CC and δ13CW showed
similar relationships with precipitation, and δ13CW
was even better related to temperature and VPD,
confirming the idea that holocellulose purification
is not strictly necessary when assessing δ13C
variation in wood. Nevertheless, for individual
tree-ring studies, the use of extracted wood may be
still needed, as resins and other extractives are
highly mobile, and would add noise to the yearly
and/or seasonal climatic signal present in cellulose
and lignin. Regarding δ18O, only holocellulose
offered a reliable climatic inference. Remarkably,
although the isotopic signal of δ18OR was
apparently lost, δ18OC still provided independent
information to that reflected in δ13C. This was
already evidenced by the poor relationship
observed between δ18O and δ13C (Table 2.3, Fig.
2.2), further suggesting that the sources of
variation for both isotopes differ partially. Hence,
δ13C variability can be mostly explained by the
stomatal regulation of CO2 fluxes into the leaf,
integrating any environmental variable related to
stomatal conductance, e.g. water availability or
VPD (Farquhar et al. 1982; Ferrio et al. 2003b).
However, it is also affected by changes in
photosynthetic activity derived from irradiance or
nutritional stresses, among others (Ferrio et al.
2003b; Livingston et al. 1998; Yakir and Israeli
1995). On the other hand, δ18O may also respond
to stomatal conductance (see Eq. 1), but it is not
affected by photosynthetic activity (Farquhar and
Lloyd 1993; Yakir 1992; Yakir and Israeli 1995).
Moreover, regardless of its effect on stomatal
conductance, VPD has a direct influence on leaf
water enrichment as, by definition, it is closely
related to the ea term in Eq. 1. Hence, in some
cases both isotopes are affected by the same
environmental variables (e.g. VPD) but, as the
processes underlying such relationships are
different, the information derived from each one
can still be complementary.
Table 2.5 Stepwise linear regression models to predict annual and seasonal means of climate variables from δ13C and/or δ18O in
different wood components. VPD, vapour pressure deficit; δ13CW, δ13CC, δ13C in whole wood and holocellulose, respectively;
δ18OC, δ18O in holocellulose.
Season
Annual
Precipitation (mm)
Model
r2
13
0.59***
-2646 - 139.6*δ CC
Temperature (ºC)
Model
r2
18
0.17*
0.1 + 0.46*δ OC
Spring
-572 - 31.6*δ13CC
0.45***
31.2 + 0.77*δ13CW
0.15*
Summer
-260 - 28.6*δ13CW
-10.3*δ18OC
-967 - 50.2*δ13CC
0.68***
33.6 + 0.87*δ13CW
+ 0.31*δ18OC
-1.4 + 0.54*δ18OC
0.37**
Autumn
Winter
-483 - 25.9*δ13CC
*P<0.05, **P<0.01, ***P<0.001
0.52***
0.34**
-
0.18*
-
VPD
Model
928 + 58.6*δ13CW
+ 41.6*δ18OC
684 + 41.7*δ13CW
+ 30.9*δ18OC
2264 + 110*δ13CW
+ 58.1*δ18OC
815 + 62.5*δ13CW
+ 48.4*δ18OC
-652 + 32.8*δ18OC
(Pa)
r2
0.52***
0.43**
0.44**
0.55***
0.31**
Published in Tellus Series B-Chemical and Physical Meteorology 57B:164-173 (2005) 75
2.4. Conclusions
According to our findings, δ13C in whole wood
may provide similar relationships with climatic
variables as those obtained with holocellulose, at
least when comparing long-term temporal series
from different sites. In contrast, our results point
out that, for δ18O, holocellulose purification is
indeed necessary to obtain consistent relationships
with climatic variables, at least when working with
the usual climatic ranges found in tree-ring studies.
On the other hand, δ18OC in P. halepensis was
mostly explained by variations in VPD, being
unrelated to δ18OR, confirming our hypothesis that
in a species with strong stomatal regulation the
signal of source water (δ18OR) would be lost due to
the great variability in evaporative enrichment at
the leaf level. Nevertheless, even in such cases,
climatic
information
δ18O may provide
independent from δ13C, as each one reflect in a
different way the effect of environmental variables
on stomatal conductance and transpirative fluxes.
Hence, we found poor correlations between δ18O
and δ13C, which leads to recommend the joint use
of δ18O and δ13C in environmental studies, as it
seems feasible to recover far more information
than by implementing both isotopes separately.
However, our results also suggest that the
particular adaptive properties at the species level
strongly determine the kind of information
provided by δ13C and δ18O. Thus, a careful
selection of the material of study (e.g. taking cooccurring species with contrasting physiological
performance)
would
probably
increase
considerably the environmental information
obtained from stable isotopes in tree-rings.
Acknowledgements
This work was partly supported by the CICYT project
BTE2001-3421-C02 and the EC project MENMED
(INCO-MED-ICA3-CT-2002-10022). J.P. Ferrio has a
PhD fellowship from the Generalitat de Catalunya. We
thank the I.N.M. and C.H.E. for providing
meteorological data, as well as IAEA/WMO for the
meteorological and isotopic data compiled from the
GNIP database. We also thank L. Serrano, A. Florit and
A. Vega for their help in sampling and processing
material, as well as the gentle collaboration and
suggestions from our colleagues at the Departament de
Química of Universitat de Lleida.
76
 Chapter 2. Carbon and oxygen isotope ratios in wood constituents of Pinus halepensis as indicators of climate
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78
 Chapter 2. Carbon and oxygen isotope ratios in wood constituents of Pinus halepensis as indicators of climate
Chapter 3
“… le daré verde a los pinos
y amarillo a la genista.”
Joan Manel Serrat. Mediterráneo
79
Chapter 3
J.P. FERRIO1 • N. ALONSO2 • J.B. LÓPEZ2 • J.L. ARAUS3 • J. VOLTAS1*
Carbon isotope composition of fossil charcoal reveals
aridity changes in the NW Mediterranean Basin
1
Departament de Producció Vegetal i Ciència Forestal, E.T.S.E.A - Universitat de Lleida,
Av. Rovira Roure 191, Lleida E-25198, Spain
2
Departament d’Història, Universitat de Lleida, Victor Siurana 1, Lleida E-25003, Spain
3
Unitat de Fisiologia Vegetal, Facultat de Biologia, Universitat de Barcelona,
Diagonal 645, Barcelona E-08028, Spain
*Corresponding author; e-mail: [email protected]
Abstract Although several proxies for the inference of precipitation have been proposed, evidence of
changes in aridity during the Holocene is scarce, and most is only qualitative. Moreover, precipitation
regimes show relatively poor spatial correlations and can exhibit contrasting responses to global
climate trends in different areas. Thus, there is a need to concentrate efforts at the local scale in order
to increase the spatial resolution of palaeoclimate records, specially regarding water availability in
semi-arid zones. Here we propose the analysis of carbon isotope composition (δ13C) in fossil charcoal
(routinely recovered from archaeological sites) to quantify changes in water availability in the past.
We applied this approach to reconstruct variations in aridity during the last four millennia in the Ebro
Depression (NE Iberian Peninsula). First, we studied the effect of carbonization over a range of
temperatures (300-500ºC) on the δ13C of Aleppo pine (Pinus halepensis Mill.) wood cores, collected
from nine locations in NE Iberian Peninsula with distinct water availability. Despite significant
changes in δ13C caused by carbonization, the original climatic signal of wood δ13C was well
preserved. Moreover, δ13C shifts induced by this process were successfully corrected by accounting
for variation in charcoal carbon concentration (%C). After removing the effect of carbonization, we
estimated annual precipitation (P) and the ratio between annual precipitation and evapotranspiration
(P/E) from the δ13C of fossil charcoal. In general, estimated water availability in the past was higher
than present values, indicating that latter-day (semi-arid) conditions are mostly due to recent climatic
changes. The good agreement between our findings and other evidence indicates that the analysis of
δ13C in charcoal may be useful to expand current palaeoclimatic records as it provides a
complementary (and quantitative) source of information to assess climate dynamics.
Keywords stable isotopes • palaeoclimate • Late Holocene • proxy • precipitation • water availability •
fossil wood • archaeology • Pinus halepensis • Iberian Peninsula
3.1. Introduction
Global trends in temperature during the
Holocene are well documented thanks to a variety
of long-term, high-resolution proxy records (see
Folland et al. 2001). In arid and semi-arid areas,
however, precipitation is the most relevant
environmental variable that determines the impact
of climate fluctuations on ecosystems, agriculture
and human settlements. Although several proxies
for precipitation have been proposed, most are only
qualitative, and evidence of past changes in aridity
during the Holocene is still scarce (Folland et al.
2001). Moreover, and unlike temperature,
precipitation regimes show relatively poor spatial
Submitted to Global Change Biology 81
correlations and can exhibit contrasting
associations with global climate trends in different
areas (see e.g. Rodó et al. 1997). This is
particularly evident for the Iberian Peninsula (NW
Mediterranean Basin), where climate is defined by
complex
interactions
between
Atlantic,
Mediterranean, Continental and Sub-tropical
influences. Indeed, a great spatial heterogeneity in
the response to global climatic trends has been
reported for the Iberian Peninsula both in present
climatic data (Rodó et al. 1997) and in the
palaeoenvironmental record (Davis 1994; Jalut et
al. 2000; Magny et al. 2003; Riera et al. 2004).
Therefore, to increase the spatial resolution of
palaeoclimatic records, especially regarding water
availability in semi-arid zones, supplementary
efforts should be concentrated at the local scale.
Carbon isotope composition (δ13C) in wood has
been related to several climatic variables that affect
plant water availability, and its analysis on treerings offers the possibility of high-resolution
climate records (see references in Ferrio et al.
2003b; Folland et al. 2001). However, vast areas of
the world have been largely deforested for a long
period, thus precluding extensive tree-ring records.
To achieve more accurate inferences on past
climatic conditions for these areas, complementary
sources of plant material in which to perform δ13C
analyses, such as charred plant remains recovered
at archaeological sites, could be used. This kind of
material is relatively common in the archaeological
context, and is systematically recovered and
identified as it provides information on agricultural
practices and ancient landscapes (Van Zeist and
Casparie 1984). The use of δ13C analysis in charred
crop grains to estimate water availability in early
agriculture has already been proposed (Araus and
Buxó 1993). This approach provides indirect
information on the short period in which the seed
is formed, although δ13C may be affected not only
by climatic variations but also by changes in crop
management practices (Araus et al. 1997b; Ferrio
et al. 2005). Alternatively, charred wood
(charcoal) offers the potential to integrate the
climatic signal over consecutive years; moreover,
it is not directly affected by agricultural practices.
Indirect evidence shows that δ13C in fossil charcoal
retains the original climatic signature present in the
wood (February 2000; Vernet et al. 1996).
However, the impact of carbonization on the δ13C
of wood has not yet been fully described, and the
relationship between charcoal δ13C and climate
remains unclear. To our knowledge, only one study
has addressed δ13C changes during charcoal
formation and it reported significantly lower δ13C
values for charred material compared to values for
intact wood (Jones and Chaloner 1991). The extent
of this difference varied depending on the
temperature treatment, which in turn affected the
degree of carbonization. Therefore, before
considering the use of charcoal as a palaeoclimatic
indicator, a precise estimation of the degree of
carbonization undergone by archaeological
remains is required.
Here we analyzed the effect of carbonization on
the δ13C of Aleppo pine (Pinus halepensis Mill.)
wood cores collected from nine locations in NE
Iberian Peninsula, which differed in water
availability. P. halepensis is a typically droughtavoiding, water-saving species that is highly
sensitive to changes in water availability, as an
adaptive strategy to the large inter-annual and
seasonal variability of Mediterranean climates
(Ferrio et al. 2003a; Liphschitz and Lev-Yadun
1986). Besides, previous studies on this species
have shown a strong relationship between several
indicators of water availability and δ13C values in
wood and its major components, cellulose and
lignin (Ferrio et al. 2003a; Ferrio and Voltas
2005). Our aim was to assess the degree of
preservation of these climatic signals in the δ13C of
wood charcoal, and to develop a model to account
for the effect (if any) of carbonization. For this
purpose,
we
performed
experimental
carbonizations over a range of temperatures, and
studied the variations in wood δ13C and carbon
concentration (%C). We applied the models
obtained to a series of archaeological samples from
82
 Chapter 3. Carbon isotopes in Pinus halepensis charcoal and palaeoclimate
the Mid Ebro Depression (NE Iberian Peninsula) in
order to reconstruct the evolution of aridity in this
area during the last 4000 years. This region is
among the most arid zones in Europe (annual
rainfall between 300 and 400 mm); however, it
remains unclear whether the present conditions are
due to recent environmental changes, or to a
progressive aridification which began in
prehistoric times. Palaeoenvironmental records for
the Late Holocene are still scarce for this area,
being mostly derived from pollen and
geomorphologic studies (Davis 1994; GutiérrezElorza and Peña-Monné 1998; Riera et al. 2004).
The application of δ13C analyses to charcoal might
provide complementary (and quantitative) data on
water availability in the NW Mediterranean Basin.
The information obtained will be discussed in
relation to current knowledge about past climates
in the region, and in other areas of Europe and the
Mediterranean.
Fig. 3.1 Geographical distribution of sampling locations for
reference wood cores (letters, empty circles), along with
archaeological sites where fossil charcoal was collected
(numbers, filled circles). Detailed descriptions of sampling
locations and archaeological sites are provided in Tables 3.1
and 3.2, respectively.
3.2 Materials and methods
3.2.1 Reference plant material: tree-ring
dating and sample preparation
We sampled wood cores (5.25 mm diameter) of
Pinus halepensis Mill. from nine locations in NE
Spain that covered a representative range of
environmental conditions for this species (Fig. 3.1,
Table 3.1). Although P. halepensis is also found in
areas that register up to 800 mm precipitation, we
did not include locations with average rainfalls
over 700 mm since above this value and according
to previous studies in the region (Ferrio et al.
2003a) wood δ13C for this species is mostly
insensitive to changes in water availability. We
selected two healthy, dominant trees per location,
and twin cores were taken from the south side of
each. Samples were oven-dried at 60ºC for 48h
before being polished to allow tree-ring dating.
After visual cross dating, each core was divided in
four segments. The number of tree-rings included
in the segments was variable (2-6) in order to
obtain fragments of similar size (10-20 mm
length), but all four cores per location followed
identical segment division. A total of 154 segments
were obtained (nine locations × two trees × two
cores × four segments).
3.2.2 Experimental carbonization
For all the analyses, and in order to minimize
individual tree effects, we pooled two segments
from the same period and location but from distinct
trees. Since two cores per tree were taken, for each
location we obtained four replicated wood
samples, corresponding to four distinct time slices
(overall 72 samples). Using direct pair-wise
comparisons between treatments, these replicates
allowed us to assess the effect of a range of
carbonization temperatures on δ13C. To this end,
we distributed all samples into four subsets of 18
samples each, which were then subjected to
distinct carbonization treatments. In particular, one
subset was kept as an untreated control, while the
other three were carbonized in a muffle furnace at
three maximum temperatures (300ºC, 400ºC, and
Submitted to Global Change Biology 83
Table 3.1 Main geographical and environmental variables of
sampling locations for reference wood cores. Lat., latitude;
Long., longitude; Alt., altitude a.s.l.; Pan, total annual
precipitation; Tan, mean annual temperature; P/Ean, ratio
between Pan and total annual evapotranspiration (Thornthwaite
1948).
Sampling
Location
A. Flix
B. El Grado
C. Leciñena
D. Lleida
E. Montserrat
F. Montsià
G. Oliana
H. Rasquera
I. Sos del R.C.
Lat.
41º15’N
42º10’N
41º48’N
41º37’N
41º34’N
40º40’N
42º05’N
41º01’N
42º28’N
Long. Alt. Pan Tan P/Ean
(m) (mm) (ºC)
0º32’E
40 389 15.9 0.46
0º22’E 520 486 13.7 0.64
0º28’W 415 368 15.2 0.44
0º38’E 220 351 14.7 0.44
1º47’E 450 387 15.4 0.47
0º32’E 200 597 17.7 0.66
1º18’E 480 628 13.9 0.82
0º36’E 135 471 15.4 0.59
1º15’W 360 609 13.2 0.84
500ºC). The temperatures were chosen to provide
material that varied in the degree of carbonization,
ranging from slightly charred to fully carbonized
(i.e. at the limit of total combustion). All samples
reached maximum temperature after a heating
ramp of 30 min and they were then kept in the
muffle for 30 min. To emulate conditions in which
charcoal is formed, we heated the samples under
restricted oxygen by burying them in metal
crucibles under 3 cm of sand.
3.2.3 Archaeological sites and
charcoal remains
169 charcoal remains of P. halepensis were
recovered from seven archaeological sites in the
Segre and Cinca Valleys (Mid Ebro Depression,
NE Spain, see Fig. 3.1, Table 3.2) which cover the
temporal range between the Bronze Age (ca. 2100
BCE) and Modern Age (XVIII c. CE). These sites
now display similar climatic and soil conditions,
and are characterized by a semi-arid Mediterranean
climate. The samples were collected from various
archaeological contexts, such as domestic fires,
cooking ovens, room floors and levels of rubble
from housing structures (for further details about
the sites and sampling procedures, see Alonso
1999; Alonso et al. 2002). For the sites from the
Bronze and First Iron period, radiocarbon ages
were determined at Beta Analytical Inc. (Miami,
USA) and at the Universitat de Barcelona
(Barcelona, Spain), and calibrated dating was
calculated with the software CALIB REV 4.3
(Stuiver et al. 1998). For the remaining sites, a
combination of stratigraphic and archaeological
dating was used. To recover plant remains, soil
samples were treated using a standard flotation
tank in the field with 5 mm, 2 mm and 0.5 mm
sieves. For the archaeobotanical analyses, charcoal
fragments of at least 5 mm were chosen, and
taxonomical determinations were performed on the
basis of the anatomical features of the wood.
Because of the carbonate enrichment of
archaeological sediments, we soaked each charcoal
fragment separately with HCl 6M for 24h at room
temperature to remove carbonate crusts (DeNiro
and Hastorf 1985). The fragments were then rinsed
repeatedly with distilled water. Carbonate removal
is a crucial step not only to avoid shifts in δ13C, but
also to achieve a proper determination of charcoal
%C.
3.2.4 Carbon concentration and
carbon isotope composition
All samples (reference and archaeological)
were oven-dried at 60ºC for 24 hours and milled to
fine powder for carbon isotope analyses. δ13C and
%C were determined by elemental analysis and
isotope ratio mass spectrometry (EA/IRMS) at
Isotope Services, Inc., Los Alamos, NM, USA. To
account for changes in δ13C of atmospheric CO2
(δ13Cair) during the Holocene, we calculated carbon
isotope discrimination in wood (∆13Cw) from
δ13Cair and wood δ13C (δ13Cw), as described by
Farquhar et al. (1982):
Eq. 3.1
∆13C w =
δ 13Cair − δ 13Cw
δ 13Cw
(1 +
)
1000
For extant wood, we obtained δ13Cair for the
years included in each core fragment from Francey
et al. (1999) and the CU-INSTAAR/NOAACMDL
network
for
atmospheric
CO2
measurements (ftp://ftp.cmdl.noaa.gov/ccg/co2c13/
flask/readme.html). The δ13Cair of the fragments
ranged from –7.4‰ to –7.9‰. For archaeological
samples, δ13Cair was inferred by interpolating a
range of data from the literature which covered the
temporal sample range (Eyer et al. 2005; Francey
84
 Chapter 3. Carbon isotopes in Pinus halepensis charcoal and palaeoclimate
et al. 1999; Indermühle et al. 1999; Leuenberger et
al. 1992).
3.2.5 Meteorological data
Meteorological data was supplied by the
Instituto Nacional de Meteorología and the
Confederación Hidrográfica del Ebro. We took
monthly values for precipitation and temperature
within the time period covered by each wood
sample used for calibration, and also from 1961 to
1990 for the reference values to characterize
present climate at the archaeological sites. From
temperature
and
latitude,
potential
evapotranspiration was calculated (Thornthwaite
1948). The ratio between precipitation and
potential evapotranspiration (P/E) was then
calculated as an index of water availability.
3.2.6 Statistical analyses
Experimental data were subjected to analysis of
variance (ANOVA) to determine the effect of
carbonization (fixed factor) and location (random
factor) on the δ13C and %C of wood segments.
Linear regressions and covariance analyses were
calculated to assess the degree of preservation of
the climatic signal in δ13C after carbonization, and
the relationship between %C and δ13C changes.
We fitted locally weighted least squares regression
curves (LOESS, span=0.3; (Cleveland 1979) to the
data sets to objectively summarize overall trends in
the time series of estimated climatic variables.
Unless otherwise stated, differences were
considered statistically significant when P<0.05.
Table 3.2 Main geographical and environmental variables (average of present values from the closest meteorological station),
dating and cultural period of the archaeological sites. Lat., latitude; Long., longitude; Alt., altitude a.s.l.; Pan, total annual
precipitation; Tan, mean annual temperature; P/Ean, ratio between Pan and total annual evapotranspiration (Thornthwaite 1948);
Calendar year, mean values and dating interval (2σ for 14C calibrated -cal. BCE- dates; estimated range for the rest of sites) for
each of the sets of samples studied.
Long.
Alt.
(m)
1. Minferri
41º30’N 0º45'E
240
389
13.5 0.51
2. Masada
de Ratón
41º29’N 0º22'E
130
350
14.6 0.43
3. Vincamet
41º30’N 0º21'E
110
350
4. Tozal de
los Regallos
5. El Vilot
de Montagut
6. Els Vilars
41º30’N 0º01'E
317
41º38’N 0º30'E
Archaeological
Site
7. Roques
del Sarró
8. Ciutat
de Lleida
Lat.
Pan Tan P/Ean
(mm) (ºC)
Calendar year (BCE/CE)
Mean
Interval
14.6 0.43
1890 cal. BCE
1673 cal. BCE
1262 cal. BCE
1110 cal. BCE
1062 cal. BCE
988 cal. BCE
2131-1692 cal. BCE
1879-1517 cal. BCE
1524-999 cal. BCE
1185-982 cal. BCE
1048-936 cal. BCE
1288-928 cal. BCE
359
14.9 0.44
896 cal. BCE
896 cal. BCE
250
355
14.7 0.45
41º34’N 0º56'E
300
403
14.2 0.51
888 cal. BCE
773 cal. BCE
488 BCE
1125-795 cal. BCE
801-396 cal. BCE
550-425 BCE
41º38’N 0º38'E
195
361
14.8 0.46
255 BCE
275-175 BCE
41º37’N 0º38'E
220
361
14.8 0.46
26 BCE
67 CE
200 CE
300 CE
925 CE
990 CE
1010 CE
1025 CE
1065 CE
1600 CE
1750 CE
50-1 BCE
20-100 CE
175-225 CE
8250-350 CE
900-950 CE
950-1030 CE
990-1030 CE
950-1100 CE
1030-1099 CE
1550-1650 CE
1700-1800 CE
Cultural
Period
Bronze Age
Late
Bronze Age
Late
Bronze Age
First
Iron Age
Late
Bronze Age
Iron Age
(Early Iberian)
Iron Age
(Iberian)
Roman
Middle Age
Modern Age
Submitted to Global Change Biology 85
-27
Table 3.3 Analysis of variance for the effect of carbonization
temperature (treatment) and geographic origin (location) in
carbon isotope composition (δ13C) and carbon concentration
(%C) of wood.
Source
Sum of
Squares
d.f.
δ13C
Treatment
Location
Treat. x Loc.
Residual
44.2
76.6
15.6
23.2
3
8
24
36
%C
Treatment
Location
Treat. x Loc.
Residual
11267.7
143.0
217.6
274.4
3
8
24
36
F
ratio
P
value
14.8
9.6
0.7
0.6
22.9
14.7
1.0
0.000
0.000
0.479
3755.9
17.9
9.1
7.6
492.7
2.0
1.2
0.000
0.095
0.312
Mean
Square
δ C
%C
-26
b
d
80
c
-25
a
13
δ C (‰)
3.3.1 Effect of carbonization on
wood δ13C and %C
For control (untreated) samples, δ13C showed
considerable variation, ranging from -26.14‰ to 22.63‰ (mean=-24.28‰ ±1.16; herein, mean ±
S.D.), whereas %C showed comparatively less
variability (48.2% ±0.7). The effect of distinct
levels of experimental carbonization (referred to as
treatment in Table 3.3) was significant for δ13C and
%C, although it was far stronger for the latter (the
treatment factor explained about 28% and 95% of
total variation, respectively, for δ13C and %C). The
geographic origin (location) of samples was still
the most determinant factor accounting for δ13C
variation (about 48%), while it was not significant
for %C. For both variables, the effect of
carbonization did not depend on the geographic
origin of the samples, as shown by the lack of
significant location by treatment interactions.
Overall, a significant increase in %C with higher
carbonization temperatures was observed, and also
a decrease in δ13C (Fig. 3.2). Although samples
treated at 300ºC showed generally lower δ13C
values (-24.50% ±1.12) than controls, they did not
differ significantly. Samples treated at 400ºC and
500ºC, however, showed a significantly lower
100
b
13
a
a
60
b
-24
40
-23
Carbon content (% C)
3.3 Results
20
control 300ºC 400ºC 500ºC
Fig. 3.2 Mean values and standard error for the δ13C and %C
in intact wood (control) and after carbonization at a range of
temperatures. Means with the same letter do not differ
significantly on the basis of a least significant difference test
(P<0.05).
average δ13C (-25.67‰ ±1.28 and –26.15‰ ±1.61,
respectively) than controls and samples treated at
300ºC. Furthermore, mean values and withintreatment variability in %C increased from 50.9%
±0.7 at 300ºC to 70.5% ±1.4 and 77.4% ±5.9 at
400ºC and 500ºC, respectively. Since δ13C and %C
showed similar responses to carbonization, we
performed a covariance analysis to determine
whether variation in %C could explain changes in
δ13C across treatments. After including %C as a
covariable in the ANOVA, the effect of
carbonization was no longer significant (P=0.601),
as this variable explained about 97% of δ13C
variation initially associated with temperature
treatments (results not shown).
The relationship between δ13C in wood before
and after charring was studied by pair-wise
comparisons of those replicated samples (i.e. from
the same location and time slice) that were
simultaneously tested under control and charring
conditions. Despite the changes observed in δ13C
after carbonization, the relationship between intact
wood and charcoal δ13C was strongly significant
across the three treatments (r2=0.64, P<0.001,
N=18). Following the results of the covariance
analysis, we performed a multiple linear regression
(stepwise) to predict original wood δ13C (δ13Cw)
86
 Chapter 3. Carbon isotopes in Pinus halepensis charcoal and palaeoclimate
using charcoal δ13C (δ13Cchar) and charcoal %C
(%Cchar, surrogate of the impact of carbonization
on δ13Cw) as independent variables. Both variables
entered the model, giving the following equation:
Eq. 3.2
δ C w = −0.706 × δ Cchar + 0.031× %Cchar − 8.07
13
13
r 2 =0.72, P <0.001
The outcome of this model therefore provided
estimations of original δ13Cw from measurements
performed on carbonized wood, that is, from
δ13Cchar and %Cchar. We applied this equation to
fossil material to obtain δ13Cw values directly
comparable to those of intact wood (see following
sections).
3.3.2 δ13C and %C in archaeological charcoal
Raw δ13C values in charcoal (δ13Cchar)
recovered from archaeological sites ranged from 25.39‰ to –20.85‰, with an average of –23.22‰.
Mean values per archaeological site and dating are
given in Table 3.4. The average %C in charcoals
was 59.1%, with values varying from 45.0% to
77.1%. After applying Eq. 2, δ13Cw showed an
average value of –22.64‰ (ranging from –24.38 to
Table 3.4 δ13C of wood charcoals recovered from archaeological sites, and present carbonized wood from the three sampling
locations closer to the study area. N, number of samples; δ13Cair, average δ13C in atmospheric CO2, according to literature data
(see text for details); δ13Cchar, δ13C values measured in wood charcoals; %Cchar, carbon concentration, on a dry mass basis; Estim.
δ13Cw, estimation of original wood δ13C, following Eq. 3.2; Estim. ∆13Cw, estimated carbon isotope discrimination of original
wood, calculated from δ13Cw and δ13Cair, as described in Eq. 3.1.
Archaeological
Site/Location
Calendar Year
(BCE/CE)
Archaeological
charcoal
1. Minferri
1. Minferri
2. Masada de Ratón
3. Vincamet
2. Masada de Ratón
2. Masada de Ratón
4. Tozal de los Regallos
5. El Vilot de Montagut
5. El Vilot de Montagut
6. Els Vilars
7. Roques del Sarró
8. Ciutat de Lleida
8. Ciutat de Lleida
8. Ciutat de Lleida
8. Ciutat de Lleida
8. Ciutat de Lleida
8. Ciutat de Lleida
8. Ciutat de Lleida
8. Ciutat de Lleida
8. Ciutat de Lleida
8. Ciutat de Lleida
8. Ciutat de Lleida
1890 cal. BCE
1673 cal. BCE
1262 cal. BCE
1110 cal. BCE
1062 cal. BCE
988 cal. BCE
896 cal. BCE
888 cal. BCE
773 cal. BCE
488 BCE
225 BCE
26 BCE
67 CE
200 CE
300 CE
925 CE
990 CE
1010 CE
1025 CE
1065 CE
1600 CE
1750 CE
Present
charcoal
A. Flix
C. Leciñena
D. Lleida
1985 CE
1992 CE
1988 CE
N
δ13Cair
(‰)
δ13Cchar
(‰)
%Cchar
Estim. δ13Cw
(‰)
Estim. ∆ 13Cw
(‰)
1
4
11
8
5
8
28
8
9
8
11
9
6
2
10
9
6
10
6
2
3
5
-6.37
-6.42
-6.50
-6.51
-6.52
-6.53
-6.54
-6.54
-6.54
-6.49
-6.45
-6.42
-6.40
-6.37
-6.36
-6.43
-6.42
-6.42
-6.43
-6.43
-6.31
-6.33
-23.73
-22.58 ± 0.60
-23.28 ± 0.26
-23.62 ± 0.14
-23.28 ± 0.30
-23.04 ± 0.10
-23.52 ± 0.17
-22.79 ± 0.23
-22.85 ± 0.15
-23.23 ± 0.13
-22.37 ± 0.40
-23.80 ± 0.21
-24.13 ± 0.43
-21.80 ± 0.03
-23.50 ± 0.12
-22.71 ± 0.40
-23.66 ± 0.35
-23.42 ± 0.13
-22.44 ± 0.48
-23.03 ± 0.18
-22.99 ± 0.02
-23.80 ± 0.33
46.02
47.72 ± 0.81
50.01 ± 1.09
62.57 ± 2.16
56.77 ± 3.15
52.93 ± 1.67
62.49 ± 1.26
58.77 ± 2.82
54.97 ± 2.47
57.52 ± 2.89
58.04 ± 2.07
59.75 ± 2.35
63.62 ± 4.43
59.12 ± 0.10
59.36 ± 1.94
61.22 ± 1.78
60.06 ± 2.47
61.32 ± 1.96
62.07 ± 2.14
57.54 ± 0.14
64.66 ± 0.88
66.93 ± 0.31
-23.40
-22.53 ± 0.40
-22.95 ± 0.18
-22.81 ± 0.12
-22.74 ± 0.27
-22.70 ± 0.06
-22.74 ± 0.14
-22.34 ± 0.21
-22.49 ± 0.16
-22.69 ± 0.12
-22.06 ± 0.32
-23.02 ± 0.15
-23.13 ± 0.43
-21.63 ± 0.02
-22.82 ± 0.08
-22.20 ± 0.30
-22.91 ± 0.29
-22.70 ± 0.12
-21.98 ± 0.33
-22.54 ± 0.12
-22.29 ± 0.04
-22.80 ± 0.23
17.43
16.48 ± 0.41
16.84 ± 0.18
16.67 ± 0.13
16.60 ± 0.28
16.54 ± 0.06
16.58 ± 0.14
16.16 ± 0.22
16.32 ± 0.17
16.57 ± 0.13
15.97 ± 0.34
17.00 ± 0.15
17.13 ± 0.45
15.60 ± 0.02
16.85 ± 0.08
16.14 ± 0.31
16.88 ± 0.30
16.66 ± 0.12
15.90 ± 0.34
16.48 ± 0.12
16.35 ± 0.04
16.85 ± 0.24
6
6
6
-7.60
-7.75
-7.67
-23.60 ± 0.35
-24.64 ± 0.18
-24.14 ± 0.48
63.97 ± 4.45
66.32 ± 4.84
64.57 ± 4.68
-22.75 ± 0.24
-23.41 ± 0.10
-23.11 ± 0.25
15.50 ± 0.24
16.04 ± 0.11
15.81 ± 0.27
Submitted to Global Change Biology 87
–20.86). Carbon isotope discrimination (∆13Cw),
calculated from estimated δ13Cw and δ13Cair, ranged
from 14.74‰ to 18.38‰, with an average of
16.55‰. Overall, the archaeological material
exhibited higher ∆13Cw values than samples taken
from the three locations closer to the area of study
800
Ferrio and Voltas (2005)
Carbonized wood
Pan (mm)
700
600
500
400
300
y = 120.1x - 1514
2
r = 0.79***
N=29
a)
200
1.0
0.9
P/Ean
0.8
0.7
0.6
0.5
y = 0.178x - 2.34
2
r = 0.83***
N=29
b)
0.4
0.3
15
16
17
18
19
∆ Cw (‰)
13
Fig. 3.3 Relationship between carbon isotope discrimination in
wood (∆13Cw) and either a) annual precipitation (Pan) or b) the
ratio between Pan and evapotranspiration (P/Ean). Empty
circles, intact wood values, as reported by Ferrio and Voltas
(2005); filled circles, estimated ∆13Cw from the analysis of
experimentally carbonized wood, as described in Eq. 3.2.
Error bars indicate standard errors for the means.
and used in the carbonization experiment
(average value of 15.79‰, Table 3.4). For most of
the archaeological sites, the variability in ∆13Cw
was similar to that found in present-day wood
charcoal (average within-site standard deviation of
0.71‰ and 0.61‰, for archaeological sites and
sampling locations, respectively).
3.3.3 Estimation of past water availability from
the ∆13C of archaeological charcoal
To assess the implications of δ13C analysis on
charcoals for palaeoclimatic reconstruction, we
also applied Eq. 2 to the wood segments that had
been experimentally carbonized, but for which
direct information on intact wood δ13C values was
not available. Estimates of δ13Cw (from δ13Cchar and
%Cchar) were used to calculate ∆13Cw values, which
were applied to infer climatic variation across the
reference locations (nine). No significant
relationship between mean annual temperature
(Tan) and estimated ∆13Cw was detected. On the
contrary, both total annual precipitation (Pan) and
the ratio between Pan and total evapotranspiration
(P/Ean) were consistently correlated with ∆13Cw
across locations for each temperature treatment
(r=0.61 to 0.96, P<0.05-P<0.001). Moreover,
neither the slope nor the intercept of these
relationships differed significantly between
treatments, according to an heterogeneity of slopes
ANOVA. Consequently, we combined ∆13Cw
values across treatments to obtain more robust
∆13Cw averages for each location. These values are
plotted in Fig. 3.3, together with those reported by
Ferrio and Voltas (2005) for intact wood of P.
halepensis covering a similar ecological gradient
(20 locations). The resulting relationships between
∆13Cw and either Pan or P/Ean were strongly
significant. The slope and intercept terms of the
regression lines did not differ significantly from
those reported in Ferrio and Voltas (2005) for
intact wood data.
88
 Chapter 3. Carbon isotopes in Pinus halepensis charcoal and palaeoclimate
D?
b)
H
D
H
D?
H? D
c)
D?
H
D
H
D?
H? D
d)
Estimated P/Ean
Pan changes (%)
100
75
50
25
0
-25
-50
-75
a)
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
100
75
50
25
0
-25
-50
-75
P/Ean changes (%)
Estimated Pan (mm)
800
700
600
500
400
300
200
100
2000 1500 1000 500 0 500 1000 1500 2000 2000 1500 1000 500 0 500 1000 1500 2000
cal. BCE
BCE CE
cal. BCE
BCE CE
Fig. 3.4 Estimated evolution of annual precipitation (Pan; a, c) or the ratio between Pan and evapotranspiration (P/Ean; b, d),
according to the estimated ∆13Cw values plotted in Table 3.4. Pan and P/Ean changes, % change relative to present climate data.
Trend lines depict locally weighted least squares regression curves (LOESS, span=0.3) fitted to the data. Relatively dry (D) and
humid (H) phases are indicated. Periods of strong climatic variability are highlighted (shadow areas).
Models from Fig. 3.3 were subsequently
applied to estimated ∆13Cw values of
archaeological charcoal in order to infer past trends
in water availability (Fig. 3.4a,b). Estimated Pan
ranged from 255 to 692 mm (472 mm in average),
whereas P/Ean varied between 0.28 and 0.93, with
an average value of 0.60. In both cases, estimated
values for archaeological samples were
significantly higher than the values recorded for
present times, averaged across archaeological sites
(Pan=361 mm, P/Ean=0.45). They were also higher
than estimations derived from δ13C values of
present-day wood collected in the same area (Ebro
Depression, locations A, C and D, see Table 3.4)
and experimentally carbonized (estimated Pan=381
mm and P/Ean=0.47). To avoid possible artifacts in
the comparison with present conditions arising
from differences in the geographic origin of
archaeological samples, we also expressed
estimated values as percentage of change with
respect to current climatic values at each
archaeological site (Table 3.2, Fig. 3.4c,d). Mean
values of Pan and P/Ean were predominantly above
present-day values, with an average increase of
about 31% and 34%, respectively. We detected
two main phases of high Pan and P/Ean (1500-900
BCE; 300 BCE-300CE), alternating with phases of
lower water availability, relatively closer to current
conditions (2000-1500 BCE; 900-300 BCE).
Finally, a period of strong climatic variability was
identified from the tenth to the eleventh century,
followed by relatively humid conditions from the
sixteenth to the eighteenth century.
Submitted to Global Change Biology 89
3.4 Discussion
3.4.1 Is the climatic signal of wood δ13C
preserved in charcoal?
The two major components in wood, lignin and
cellulose, differ substantially in their isotopic
composition, the former showing a lower δ13C than
the latter (Borella et al. 1998; DeNiro and Hastorf
1985; Ferrio and Voltas 2005). Moreover, cellulose
is more rapidly combusted than lignin (Beall et al.
1974; Kollmann 1955), thus causing substantial
isotopic depletion of charcoal when compared to
intact wood (Jones and Chaloner 1991).
Nevertheless, since δ13C displays similar responses
to climate in cellulose and lignin (Borella et al.
1998; Ferrio and Voltas 2005), this fractionation
during charring would not override the climatic
signal of charcoal δ13C. In fact, and despite
considerable changes in δ13C during carbonization,
our results reveal that the environmental signature
of wood δ13C is still present in charcoal. This can
be derived not only from the persistence of a
geographic signature across a range of heat
treatments, but also from the strong correlation
between δ13C in intact and carbonized wood.
Indeed, we obtained a model to estimate the
original δ13C of wood (i.e. before carbonization)
from charcoal δ13C, including %C as an indicator
of the degree of carbonization. Using this model,
we found consistent relationships between ∆13C of
carbonized wood (corrected for the effect of
carbonization) and climatic variables, and these
relationships did not differ significantly from those
previously reported for intact wood of P.
halepensis (Ferrio et al. 2003a; Ferrio and Voltas
2005). Our methodology can be easily
implemented in δ13C studies in charcoal, as it does
not require further measurements (%C is routinely
included in carbon isotope analyses). Nevertheless,
as the δ13C changes observed during carbonization
are mostly attributable to differential combustion
of lignin and cellulose, and their amounts can
differ among species (Kollmann 1955), this
particular correction model might not be directly
applicable to other species, mainly non-conifers.
3.4.2 %C in charcoal as indicator of
carbonization level
As expected, %C had a low range of variation
in intact wood, even in trees grown under
contrasting environmental conditions. Indeed, this
variable shows a rather constant value of around
50%, regardless of the species, environmental
conditions or type of wood (Kollmann 1955). As
%C increases progressively during charring, it is a
good indicator of the degree of carbonization,
which can be potentially useful to correct d13C
shifts related to this process. However, it should be
noted that the range of variability of %C increased
considerably with increasing temperature levels.
The most probable explanation for this effect
would be that the heat released during combustion
further increased the effective temperature of
carbonization, particularly at the highest
temperatures. This process, known as selfcombustion, is enhanced by resins and other
volatiles, and thus may differ from one piece of
wood to another (Kollmann 1955). Similarly, this
process would explain the greater spread of d13C
values for the highest temperature treatment.
The average %C in archaeological samples
(59.1%) indicates that most of the samples reached
intermediate carbonization levels between the
300ºC and 400ºC treatments. This observation is
consistent with evidence from anatomical studies
and spectral measurements (Guo and Bustin 1998;
Jones and Chaloner 1991), thereby indicating that
most of the fossil charcoal was formed between ca.
350ºC and ca. 450ºC. This “optimal” temperature
range is probably the result of the two critical
requirements for the preservation of fossil plant
remains. They must become inert enough to
prevent microbial decomposition, while retaining
some physical resistance to avoid destruction
during burial and post-burial processes, including
recovery (Ferrio et al. 2004; Jones and Chaloner
1991; Van Zeist and Casparie 1984; Wright 2003).
90
 Chapter 3. Carbon isotopes in Pinus halepensis charcoal and palaeoclimate
3.4.3 Changes in aridity during the last 4000
years in the NW Mediterranean Basin
In spite of the aforementioned constraints, our
approach provided a robust quantification of
climatic variables directly associated with water
availability, such as Pan and P/Ean. This
methodology makes the comparison between past
and present-day conditions easier than the most
common
qualitative
or
semi-quantitative
approaches (see references in Folland et al. 2001).
In our study, estimated Pan and P/Ean from the
analysis of charcoal δ13C were significantly higher
in the past than in present times, and showed an
increase over 30% in relation to current values.
These results were supported by δ13C analysis of
experimentally carbonized wood from three
reference locations (A, C and D in Table 3.1), as
these sites provided significantly lower estimations
of Pan and P/Ean than the average for archaeological
samples. Moreover, the good agreement between
recorded and estimated values across the three
reference locations further supports the validity of
our models (estimated Pan=381 mm and
P/Ean=0.47, climatic data Pan=379 mm and
P/Ean=0.48).
On the basis of these findings, present-day
semi-arid conditions in the study area may be
mostly due to recent climatic shifts towards a
harsher
climate,
probably
enhanced
by
anthropogenic disturbance. A considerable
decrease in precipitation in NW Mediterranean has
been reported after 1850, as shown by tree-ring
analyses (Creus et al. 1996), lake-level records
(Riera et al. 2004) and documentary studies
(Barriendos and Martín-Vide 1998). In addition to
a fall in precipitation, the global increase in
temperature since the mid XIXth century have
augmented plant water demand, and, consequently,
water stress, throughout the Mediterranean Region
(Creus et al. 1996; Folland et al. 2001).In addition
to the overall differences observed between past
and present-day values, two main phases of greater
water availability alternating with drier periods
were detected. The first humid phase (1500-900B
CE) gave significantly higher averages of Pan and
P/Ean than those for the present (increase of 36%
and 42%, respectively). This humid period
coincided with the interphase between two
aridification
periods
(2350-1450BCE
and
900BCE-220 CE) as defined by Jalut et al. (2000)
for NW Mediterranean on the basis of a transect of
pollen analyses. Because of the small number of
samples available, the relatively arid episode
described by Jalut et al. (2000) around 23501450BCE was not well characterized in our data.
Nevertheless, we found a trend towards lower
water availability from 2000BCE to 1500BCE
(when compared to the following period) although,
on average, the Pan and P/Ean estimated for this
period were still higher than present-day values.
The most consistent arid phase within the
timeframe of this study corresponded to the period
900-300BCE. Conversely, even in this phase, our
estimates of water availability were significantly
higher than for present times (about 21%). This
period was followed by a recovery of water
availability between 300 BCE and 300 CE, with
average Pan and P/Ean about 33% and 36% higher
than present-day values. The analysis of δ13C in
charcoals from a Mediterranean deciduous oak
(Quercus pubescens W.) performed by Vernet et
al. (1996) in Southern France also indicated a
period of lower water availability ca. 3000-2700
BP, a subsequent recovery ca. 2200-1600 BP, and
a declining trend onwards until present times.
Estimated water inputs of archaeological cereal
crops (wheat and barley) grown in NW
Mediterranean (Araus and Buxó 1993) are also
consistent with these general trends. Furthermore,
pollen analyses performed at the archaeological
site of El Vilot de Montagut (Alonso et al. 2002)
reveal an increase in aridity at the beginning of the
3rd millennium, which is in agreement with the low
∆13C values found for this site. Likewise, the
Submitted to Global Change Biology 91
humid phase between 300 BCE and 300 CE was
also confirmed by pollen data from the city of
Lleida (0-200 CE), which indicated a relatively wet
climate (Riera S., unpublished results). Similarly,
lower groundwater fluxes from 500 to 100 BCE
than between 100 BCE and 200 CE have been
reported (Magny and Richard 1992). Some studies,
however, provide apparently contradictory results
for the arid period 900-300 BCE. Geomorphologic
studies throughout the Ebro Basin (GutiérrezElorza and Peña-Monné 1998) report a generalized
increase in sediment accumulation on slope
deposits around 1000-400 BCE, which was
interpreted as an increase in vegetation cover, and
a colder/wetter climate. Gribbin and Lamb (1978)
had previously described a decrease in temperature
of about 2ºC for this period, which was further
supported by an expansion of the glaciers, and
evidence of a colder climate in North and Central
Europe (see e.g. Magny et al. 2003). These
findings are partly supported by vegetation
reconstructions (based on charcoal analyses) from
the Cinca and Segre Valleys (Alonso 1999), which
show the presence of mixed forests of holm oaks,
pines and deciduous oaks (i.e. colder/wetter
climate than today). The most probable
explanation for such divergent results might be
found in the distinct physiological responses of P.
halepensis to temperature and water regimes. An
abrupt decrease in winter temperatures would have
shortened the growing cycle of this species, as it is
sensitive to cold and can virtually arrest growth
during the cold season (Liphschitz and Lev-Yadun
1986). In addition, although lower temperatures are
expected to reduce water demand in summer, they
might also have reduced the incidence of
convective storms (and thus water availability)
from late spring to early autumn (Gribbin and
Lamb 1978). Indeed, ∆13C analyses of cereal grains
(Araus et al. 1997a; Araus and Buxó 1993)
indicate that water inputs during grain filling (late
spring) decreased significantly during this period
in NW Mediterranean. Moreover, in the nearby
Monegros region, charcoal studies show a decrease
in vegetation cover, and also an increase in
continentality, thereby leading to the disappearance
of cold-sensitive species (Alonso 1999).
Consequently, a combination of colder/wetter
winters, but drier summers, might have caused an
overall increase in water stress for P. halepensis
during this period. This hypothesis would be fully
compatible with a reduction of soil erosion (,
enhanced by the absence of convective storms,
without discarding an increase in vegetation cover,
driven by cold-resistant species with deep-root
systems to exploit winter-fed water reservoirs, e.g.
Mediterranean oaks (Alonso 1999). Expanding
∆13C analyses to these species might help to
elucidate the origin of these contrasting results, and
provide useful information on the seasonality of
precipitation events.
We identified three periods of strong climatic
variability (shaded areas in Fig. 3.4: 900-800 BCE;
275-175 BCE; 900-1100 CE), the first two
coinciding with the transition between the
aforementioned phases of higher and lower water
availability. According to our data, the third period
was characterized by relatively low and unstable
Pan and P/Ean values (but still 26-28% above
current values). Because of the lack of samples
dating immediately before and after the 900-1100
CE, we cannot attribute the great variability of this
period to a “transition effect”. This phase coincides
with the Medieval Warm Period (Gribbin and
Lamb 1978), dated between 1000 and 1200 CE,
and characterized by an overall increase in
temperature. According to those authors, an
increase in summer temperatures would cause
higher water deficits, and the importance of
convective storms would be increased, which is
consistent with the dry and unstable climatic
conditions reported here. Pollen data from the city
of Lleida also indicate drier conditions in this
period than at the beginning of the 1st Millennium
CE (Riera S., unpublished results). Documentary
92
 Chapter 3. Carbon isotopes in Pinus halepensis charcoal and palaeoclimate
studies for this period reflect a climate with strong
interannual contrasts, alternating drought periods
with catastrophic floods (Gargallo 1989).
Furthermore, soil erosion increased around 4001200 CE ((Gutiérrez-Elorza and Peña-Monné
1998), which is consistent with a more arid and
variable climate. Our analyses revealed a
subsequent recovery in water availabity from the
XVIth to the XVIIIth century (35-38% over
current values), although, because of the small
number of samples available, these results are not
conclusive. Nevertheless, this period coincides
with the Little Ice Age (1450-1850 CE), which is
characterized by strong climatic variability
(Barriendos and Martín-Vide 1998; Creus et al.
1996; Riera et al. 2004), lower mean temperatures
(Creus et al. 1996; Gribbin and Lamb 1978),
reduced erosion (Gutiérrez-Elorza and PeñaMonné 1998) and increased lake levels (Riera et
al. 2004).
3.5 Conclusions
Our experimental data demonstrates that the
climatic signal of wood δ13C is preserved after
carbonization, and that shifts in δ13C caused by this
process can be successfully removed. Moreover,
the generally good agreement between our findings
and
evidence
provided
by
several
palaeoenvironmental data from NW Mediterranean
supports the usefulness of δ13C analysis in wood
charcoal to expand current palaeoclimatic records,
especially in dry areas where other records (e.g.
lake sediments, ice-cores, tree-rings) are relatively
scarce. Even in regions where long palaeoclimatic
records are available, δ13C in charcoal might
provide a complementary (and quantitative) source
of information, in order to achieve a greater
understanding of past climatic dynamics. The
divergences between our results and some
palaeoenvironmental evidence, however, highlight
the complexity of combining information from
distinct sources. Nevertheless, interdisciplinarity is
an essential prerequisite for palaeoenvironmental
studies, as each proxy has its own constraints,
which can be overcome only by considering
alternative inferential sources. In this regard, the
particular physiological characteristics of the
species under investigation must be considered to
ensure a proper interpretation of δ13C trends in
charcoal.
Acknowledgements
This work was partly supported by the CICYT project
BTE2001-3421-C02 and the EC project MENMED
(INCO-MED-ICA3-CT-2002-10022). We thank Maite
Ros and Raquel Piqué for performing charcoal
determinations, Alicia Florit, Anna Vega and Jaime
Coello for assistance in sampling preparation, and
Francisco Rodriguez for useful suggestions regarding
statistics. We also thank the excavation teams from
Masada de Ratón (J. Rey), Tozal de los Regallos (S.
Melguizo), the Servei Municipal d’Arqueologia de
Lleida, as well as the staff from the Grup d’Investigació
Prehistòrica for their work and financial support for
sampling dating. J.P. Ferrio has a Ph.D. fellowship from
the Generalitat de Catalunya.
Submitted to Global Change Biology 93
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Chapter 4
“Le blé pour moi est inutile. Les champs de blé ne
me rappellent rien. Et ça, c’est triste!”
Antoine de Saint Exupérry. Le Petit Prince
97
Chapter 4
J.P. FERRIO1 • J.L. ARAUS 2 • R. BUXO3 • J. VOLTAS1 • J. BORT2
Water management practices and climate in ancient agriculture:
inference from the stable isotope composition
of archaeobotanical remains
1
Departament de Producció Vegetal i Ciencia Forestal, E.T.S.E.A-Universitat de Lleida, Av.
Rovira Roure, 191, Lleida E-25198, Spain
2
Unitat de Fisiologia Vegetal, Departament de Biologia Vegetal, Facultat de Biologia,
Universitat de Barcelona, Av. Diagonal, 645, Barcelona E-08028, Spain
3
Museu d’Arqueologia de Catalunya, C. Pedret, 95, Girona E-17007, Spain
*Corresponding author; e-mail: [email protected]
Abstract Carbon isotope discrimination (∆13C) in charred grains from archaeological sites provides
reliable information about water availability of ancient crops. However, as cereals are cultivated
plants, they may reflect not only climatic fluctuations, but also the effect on water status of certain
agronomic practices, such as sowing in naturally wet soils or irrigation. In this work, we propose a
methodological approach to combine ∆13C data from different plant species, in order to discriminate
between climate-derived and anthropogenic effects on ancient crops. We updated previous models to
estimate water inputs from ∆13C of cereal grains (Hordeum vulgare and Triticum aestivum/durum),
and we applied them to compiled literature data from several archaeological sites, including samples
from Neolithic to present-day in NE and SE Spain, as well as from the Neolithic site of Tell Halula
(NW Syria). We found an important decrease in water availability from Neolithic to present-time in
the three areas of study, specially clear for the two driest areas (SE Spain and NW Syria). Potential
differences in water management practices between wheat and barley, as well as between cereal and
legume crops (Vicia faba and Lens culinaris), were also discussed, based on the comparison of ∆13C
values across several archaeological sites.
Keywords Carbon isotope discrimination (∆13C) • wheat • barley • legumes •
Middle Euphrates • Iberian Peninsula
4.1. Introduction
Improving our knowledge about the way early
farmers reacted to environmental changes might
assist to face current conflicts resulting from water
shortage in different areas of the world. Up to now,
the most evident way to get insight into ancient
water management methods is the study of
archaeological structures related to water uptake or
distribution. However, this methodology is limited
to relatively advanced societies, and does not give
any information about the actual results of such
management on crops performance. Several
indirect methods have been proposed to determine
the water status of ancient crops, such as the size
of charred seeds (Ferrio et al. 2004; Helbaek
1960), the analysis of phytoliths (Rosen and
Weiner 1994), or the study of weed flora (Jones et
al. 1995). However, none of these methods is
conclusive, and some of them, such as phytolith
analyses, are only applicable to certain crops.
Published in Vegetation History and Archaeobotany 14:00-00 (2005) 99
Carbon isotope composition (δ13C) in C3 plant
tissues constitutes an integrated record of the ratio
of intercellular to atmospheric concentration of
CO2 during the period in which the carbon was
fixed, and thus reflects the balance between carbon
fixation and stomatal conductance. Plants typically
react against a decrease in water availability
through stomata closure and, thus, δ13C from plant
tissues is a good indicator of the water status
during the time these tissues were formed (see
Farquhar et al. 1989 for further details on carbon
isotope theory). Many studies under growthchamber and field conditions have shown that
plants developed under water stress produced
leaves with higher (i.e. less negative) δ13C values
(see references in Ferrio et al. 2003b; Hubick et al.
1993). According to these findings, a significant
relationship between δ13C and environmental
parameters related to water availability would be
expected. Although most of the basic studies on
δ13C and plant water relations have been performed
on leaf material (as this is the tissue directly
involved in photosynthesis), further works has
shown that similar relationships can be established
using other plant tissues such as seeds or wood.
Wood δ13C has been related to changes in
various climatic variables, including humidity
(Saurer and Siegenthaler 1989; Stuiver and
Braziunas 1987), precipitation (Ferrio et al. 2003a;
Warren et al. 2001) and water pressure deficit
(Ferrio and Voltas 2005; Korol et al. 1999), among
others. Although most of these works were limited
to the last 100 or 200 years, such relationships
have been recently extended over longer tree-ring
chronologies, allowing high resolution climatic
reconstructions back to ca. 11000 B.P. (see
references in Heaton 1999). However, these studies
are limited to a few world regions where such
long-term chronologies are available. Even though
they provide important keys for the understanding
of global climate changes, it would be helpful to
find alternative sources of information to refine
climatic reconstructions at the local scale. In this
context, Araus and Buxó (1993) proposed the use
of δ13C in charred grains from archaeological sites
to get insight into the environmental conditions in
early agriculture. This approach has been
subsequently improved to allow the quantification
of cereal water inputs (Araus et al. 1997a; Araus et
al. 1999a) and crop yields (Araus et al. 1999b;
Araus et al. 2003a). These works are based on the
comparison with modern reference material, since
δ13C of seeds shows negligible changes during
carbonisation, and thus the original isotopic signal
is well preserved in charred remains (Araus et al.
1997b; Marino and DeNiro 1987). The impact of
carbonisation on the δ13C of wood has not yet been
fully described, but indirect evidence shows that
δ13C in fossil charcoal also retains the original
climatic signature of wood (February 2000; VanKlinken et al. 1994; Vernet et al. 1996).
In this context, it is necessary to find some
additional clues to interpret the inferences of
ancient crop conditions derived from δ13C
analyses. Indeed, crop water availability, and thus
δ13C values in cereal crops, may be affected not
only by climatic variations, but also by changes in
crop management, such as irrigation practices
(Araus et al. 1997b). To solve this question, one
possibility is to compare δ13C values found in
cultivated plants with those from wild species (e.g.
trees), which are not directly affected by
agricultural practices. On the other hand,
comparing the results from different crops can
provide
evidence about selective water
management. In this work, we propose the use of
combined δ13C analyses of different plant species,
as a way to get further information from isotope
data, with the aim to discriminate between climatederived and anthropogenic effects on crop water
status, as well as to distinguish different strategies
of water management.
100
 Chapter 4. Water management practices and climate in ancient agriculture
4.2. Materials and Methods
4.2.1. Review on δ13C data from
archaeological grains
We compiled reported δ13C values from several
archaeological sites in NE and SE Spain (Araus et
al. 1997b; Araus and Buxó 1993) and in the
Neolithic site of Tell Halula, in NW Syria (Araus
et al. 1999a; Ferrio et al. 2003b). The dataset (see
Table 4.1) included δ13C values from the cereals
hulled barley (Hordeum vulgare), naked wheat
(Triticum durum/aestivum) and the legumes faba
bean (Vicia faba minor) and lentil (Lens culinaris).
4.2.2. Determination of ∆13C values
To account for changes in δ13C of atmospheric
CO2 (δ13Cair) during the Holocene, we calculated
plant ∆13C from δ13Cair and plant carbon isotope
composition (δ13Cp), as described by Farquhar et
al. (1982):
Eq. 4.1
∆13C w =
δ Cair − δ Cw
δ 13Cw
(1 +
)
1000
13
13
δ13Cair was inferred by interpolating a range of
data from ice-core records covering the whole
Holocene (Eyer et al. 2004; Francey et al. 1999;
Indermühle et al. 1999; Leuenberger et al. 1992).
We recalculated ∆13C for all the archaeological
data compiled, using these updated estimations of
past δ13Cair.
4.2.3. Reference ∆13C values in present crops
Average values of ∆13C in modern cereals and
legumes were compiled from the literature (Araus
et al. 1997b; Araus et al. 2003b; Ferrio et al. 2001;
Voltas et al. 1999). The dataset included data from
barley (H. vulgare), wheat (T. aestivum and T.
durum combined) and faba bean (V. faba) grown in
NE Spain, SE Spain and NW Syria, either under
irrigated or rainfed conditions (see Table 4.2). We
took advantage of the additional compiled data to
update the model developed by Araus et al.
(1999a) to estimate crop water inputs from ∆13C in
wheat grains.
4.2.4. Meteorological data
In order to enable comparisons among data
from different geographic origins, we used as
reference values total precipitation from the second
half of April to the end of May, coinciding,
approximately, with the average grain filling
period across the studied areas. Meteorological
data was supplied by the Instituto Nacional de
Meteorología, the International Center for
Agricultural Research in Dry Areas (ICARDA)
and the Confederación Hidrográfica del Ebro.
4.2.5. Statistical analysis
Heterogeneity of slopes ANOVA was
performed to determine the differences between
barley and wheat in their relationship between
∆13C and water inputs. δ13Cair data from different
sources was interpolated by fitting locally
weighted least squares (LOESS) regression curves
(Cleveland 1979). Due to the far greater time
resolution of δ13Cair data available since ca. 1800
A.D., we performed two separate LOESS
regressions, one including data from 16100 cal.
B.C. to 1800 A.D. (span = 0.1) and the other for
the period 1798-1996 A.D. (span=0.4). The
resulting curves were hereafter used as a single
one, since they gave nearly identical estimations at
the jointing point, around 1800 A.D. (end of the
first curve: δ13Cair(1800)= -6.402; beginning of the
second curve: δ13Cair(1798)= -6.406). A linear
regression between ∆13C of archaeological grains
of wheat and barley across sites were used to
determine the degree of agreement between the
two species, as well as to find potential outliers,
according to the 95% confidence interval of the
regression line.
Published in Vegetation History and Archaeobotany 14:00-00 (2005) 101
102
 Chapter 4. Water management practices and climate in ancient agriculture
Published in Vegetation History and Archaeobotany 14:00-00 (2005) 103
Table 4.2 Average values of carbon isotope discrimination (∆13C) in wheat, barley and faba bean grains grown under rainfed and
irrigated conditions in the three geographic areas studied. Mean plus standard error are indicated, as well as the number of field
trials analysed, between parentheses. For the rainfed trials, we include also water inputs (WI) during grain filling (estimated from
∆13C values) and, for comparison, accumulated rainfall from the second half of April to the end of May (P ½ A+ M). ∆13C values
compiled from Araus et al. (1997b; 2003b), Ferrio et al. (2001) and Voltas et al. (1999).
Rainfed/
irrigated
NE Spain
Rainfed
Irrigated
Rainfed
Irrigated
Rainfed
Irrigated
SE Spain
NW Syria
H. vulgare
16.4 ± 0.5 (9)
19.0 ± 0.4 (5)
15.1 ± 0.5 (9)
18.7 (1)
14.6 ± 0.6 (2)
∆ 13C (‰)
T. aest/durum
15.4 ± 0.9 (3)
17.9 ± 0.5 (3)
14.9 ± 1.0 (7)
16.9 ± 0.1 (6)
14.3 ± 0.5 (2)
16.8 ± 0.1 (2)
WI (mm)
H.vulgare T. aest/durum
V. faba
14.9 (1)
16.7 ± 0.6 (3)
4.3. Results and Discussion
4.3.1. Modelling water inputs from ∆13C in
grains
The models relating ∆13C in cereal grains and
water inputs during grain filling are plotted in Fig.
4.1. (Araus et al. 1997a; Araus et al. 1999a)
already reported strong positive relationships
between water inputs and ∆13C values from barley
(Hordeum vulgare) and wheat (Triticum
durum/aestivum) grains across a range of
environmental conditions. After adding data from
six wheat trials located at the Iberian Peninsula,
both
species
provided
nearly
identical
relationships, indicating that, despite their
differences in growth cycle and ecological
preferences, they show similar physiological
responses to water availability. Nevertheless,
wheat ∆13C values tended to be smaller than those
of barley, especially at the driest environments, in
agreement with the greater sensitivity of wheat to
water stress. On the other hand, it should be noted
that, in both cases, the observed relationship was
not lineal, and thus ∆13C was more sensitive to
water availability under harsher conditions. This
can be explained by the fact that the main factor
relating ∆13C with water availability is the ratio
between intercellular and atmospheric CO2
concentrations, which is supposed to reach its
P ½ A+M
(mm)
82 ± 14
62 ± 20
70
51 ± 10
52 ± 15
59
42 ± 9
41 ± 8
35
maximum value in non-stressed plants. Thus,
under near-optimum crop water status, no further
increments in this variable, and thus on ∆13C,
would be expected (Farquhar et al. 1989; Lambers
et al. 1998).
20
19
13
Grain ∆ C(‰)
Geographic
area
18
17
16
15
14
Wheat
Barley
13
12
0
50
100
150
200
250
Water Inputs (mm)
Fig. 4.1 Relationship between water inputs (WI, rainfall plus
irrigation if applied) during grain filling and ∆13C of barley
(Hordeum vulgare, empty circles) and wheat (Triticum
aestivum and T. durum, filled circles) grains. Data from Araus
et al. (1997a) for barley and from Araus et al. (1999a; 2003b)
for wheat; ∆barley= 9.99 + 1.52*ln (WI), r2=0.73, P<0.001,
N=25; ∆wheat= 8.50 + 1.78*ln (WI), r2=0.73, P<0.001, N=22.
104
 Chapter 4. Water management practices and climate in ancient agriculture
∆13C(‰)
Water inputs (mm)
Reconstruction of past climate changes in the
Mediterranean Basin
A case study of the application of
archaeological plant remains to the analysis of
climate fluctuations is shown in Fig. 4.2. The
evolution of δ13Cair during the Holocene, as
inferred from ice-core records, is shown at the top
of Fig. 4.2a. Average δ13C values in cereal grains
(wheat and barley combined) are plotted at the
bottom of Fig. 4.2a. The data here presented was
compiled from the archaeological sites described in
Table 4.1, as well as from present agronomic trials
(see Table 4.2). ∆13C values were then recalculated
from the updated estimations of δ13Cair and
measured δ13C in grains, as described in equation 1
(Fig. 4.2b). Finally, by applying the modelled
relationship between ∆13C and water inputs (Fig.
4.1), it was possible to estimate water availability
in the past from ∆13C of ancient grain samples
(Fig. 4.2c). We observed an important decrease in
water availability from Neolithic to present-time in
the three regions of study, specially clear for the
two driest areas (SE Spain and NW Syria). These
results indicate that, during early agriculture,
cereals were cultivated under much better water
status than that expected from present-day
(rainfed) conditions in the same areas. This finding
is in agreement with archaeobotanical evidence
supporting the possibility that environmental
conditions during early agriculture were cooler and
moister than today, both in the Near East (Harlan
1998; Willcox 1996) and the Iberian Peninsula
(Vernet 1990). The origin of such increase in
aridity appears to be relatively recent, and has been
probably enhanced by human activities. Indeed,
several
palaeoenvironmental records have
evidenced a considerable decrease in precipitation
in the Mediterranean since mid XIXth century,
along with a global rise in temperature (Barriendos
and Martín-Vide 1998; Creus et al. 1996; Folland
et al. 2001; Riera et al. 2004).
δ13C(‰)
4.3.2. Applications of ∆13C to fossil plant
remains
-6
-7
-8
-21
-22
-23
-24
-25
19
18
17
16
15
14
250
a)
b)
c)
NE Spain
SE Spain
NW Syria
200
150
100
50
10
8
6
4
2
0
Calibrated Age (Ky B.P.)
Fig. 4.2 Estimation of past water inputs from carbon isotope
discrimination (∆13C) of archaeological cereal grains
(Hordeum vulgare and Triticum aestivum/durum combined)
collected from archaeological sites located in three areas of the
Mediterranean Basin (NE Spain, SE Spain and NW Syria, see
Table 4.1). (a) Evolution of the isotopic composition (δ13C) in
atmospheric CO2 (top) and archaeological cereal grains
(bottom). Both variables (air and plant δ13C) are required to
calculate ∆13C of grains (b), as described in Equation 4.1. (c)
Evolution of cereal water inputs (estimated from ∆13C) from
Neolithic to present times in the areas studied.
Published in Vegetation History and Archaeobotany 14:00-00 (2005) 105
further check the climatic effect on crop species,
we are currently determining the carbon isotope
signature of charcoals from the same sites where
cereal grains were collected (Ferrio, Alonso,
López, Araus and Voltas, submitted manuscript).
By comparing the results of forest and cultivated
species co-occurring in the same sites, it may be
possible to estimate more accurately the relative
influence of human activities on the water status of
crops.
a)
150
100
50
250
Past WI (mm)
Present WI (mm)
21
b)
20
200
19
150
18
100
17
50
Vernet et al. (1996)
0
8
7
6
5
4
3
2
1
16
13
Wood ∆ C(‰)
Past WI (mm)
200
Relative WI (%)
Is it feasible to discriminate between climatic
and anthropogenic effects on ∆13C?
As cereals are cultivated plants, they may
reflect not only climatic fluctuations, but also the
effect on water status of certain agronomic
practices, such as sowing in naturally wet soils
(Hillman 1996) or irrigation (Helbaek 1960). This
might be the case, for example, in some sites from
NE and SE Spain, where estimated water inputs
fell over the expected values for rainfed
cultivation, even considering a wetter climate in
the area (e.g. Montou and Cova 120, in NE Spain;
Puente Tablas, Peñalosa, Cuesta del Negro, Cerro
de la Virgen and Los Millares, in SE Spain). A
possible way to discriminate between natural and
anthropogenic changes in plant water status is to
compare the carbon isotope signature of cultivated
species with that of wild species, such as forest
trees. This is exemplified in Fig. 4.3, where we
compared the estimated water inputs for barley and
wheat grains collected in NE Spain with ∆13C
values in wood charcoals from SE France,
calculated from δa (see Fig. 4.2a) and wood δ13C
data reported by Vernet et al. (1996) for Quercus
humilis (deciduous oak) and Juniperus sp.
(juniper). As these two areas are close to each
other and display similar climate regimes, we
would expect to find common trends in both
studies if climate were the main factor determining
crop water status. As in cereals from NE Spain,
Vernet et al. (1996) found evidence of higher
water availability in the past than in the presentday. Moreover, both studies displayed relatively
dry episodes (around 7000 B.P., 5000 B.P., 3000
B.P. and 1000 B.P.), alternating with more humid
periods (around 6000 B.P. and 4000 B.P.), and
concluding with a new decrease in water
availability during the last millennium. The
relatively good agreement between the results
obtained for cereals and forest species suggests
that most of the observed changes in crops may be
explained by climatic conditions. As a way to
15
0
Calibrated Age (Ky B.P.)
Fig. 4.3 (a) Evolution of estimated water inputs (WI) in
archaeological cereal grains (Hordeum vulgare and Triticum
aestivum/durum combined) collected in archaeological sites
from NE Spain, compared with average present values in the
sites (as described in Table 4.1). (b) Evolution of WI, as a
percentage respect current values. For reference, a horizontal
line is plotted at WI=100% of present values. The dotted curve
indicates the evolution of ∆13C in wood charcoals of deciduous
oak (Quecus humilis) and juniper (Juniperus sp.), collected at
several archaeological sites from SE France. Charcoal ∆13C
values were calculated from wood δ13C (Vernet et al. 1996)
and air δ13C, as described in Equation 4.1.
106
 Chapter 4. Water management practices and climate in ancient agriculture
Barley ∆ C(‰)
13
14
20
15
16
17
18
19
20
a)
Wheat ∆13C(‰)
19
18
17
16
∆wheat=4.50+0.72*∆barley
15
2
r =0.65***
14
Legumes ∆13C(‰)
An alternative approach to detect selective
water management strategies is based on the
comparison of crop water status among different
crop species grown in the same area. This is shown
in Fig. 4.4, putting together all the data described
in Table 4.1. Hence, we compared the relationship
across sites between ∆13C of barley and wheat
grains (Fig. 4.4a) and that between ∆13C of cereals
(barley and wheat combined) and either faba bean
(Vicia faba minor) or lentil (Lens culinaris) (Fig.
4.4b). We found that ∆13C values in barley and
wheat grains were well correlated, showing similar
trends across the sites. This suggests that both
cereals were grown under similar conditions, and
that observed fluctuations were mostly due to
common (e.g. climatic) factors affecting their
water status. In contrast, values of ∆13C for faba
beans and lentils were unrelated to those of cereal
grains, being also significantly higher (about 1‰
in average). Differences in ∆13C between cereals
and grain legumes could be explained (at least in
part) in terms of differences in grown pattern.
Whereas cereals are determinate plants, producing
all ears at one time, and grain growth coincides
with the onset of drought in the Mediterranean
region, legumes are indeterminate plants,
producing successive pods throughout the crop
cycle (generally from March to June). Therefore,
for faba beans and lentils, most seeds may develop
under a higher water status. On the other hand, the
possibility that legumes were irrigated, even when
cereals were cultivated under rainfed conditions,
cannot be discarded. Indeed, we found that ∆13C of
archaeological faba beans were closer to those
measured in present-day irrigated crops near the
sites than to those of rainfed crops (see Table 4.2).
On the other hand, the frequency of legume seeds
found in the archaeological contexts studied was
considerably lower than that of cereal grains. In
spite of the uncertainties associated to the uneven
20
19
b)
18
17
16
Lentil
Faba bean
15
14
14
15
16
17
18
19
Wheat and Barley ∆ C(‰)
13
20
Fig. 4.4 Relationship between ∆13C values of hulled barley
(Hordeum vulgare) and either naked wheat (Triticum
aestivum/durum) or faba bean (Vicia faba minor) across all the
archaeological sites from Table 4.1. (a) Detection of potential
outliers for the relationship between wheat and barley, taking
present data for comparison. Empty symbols, present-day
samples, used for reference (see Table 2); filled symbols,
archaeological samples; dotted lines, 95% confidence intervals
for the regression line; dashed line, 1:1 reference line. Origin
of the samples: circles, NE Spain; triangles, SE Spain;
squares, NW Syria. The encircled points correspond to
present-day values for irrigated wheat, against rainfed barley;
the points surrounded by hexagons are “outlier”
archaeological sites where wheat displayed higher ∆13C
values, probably indicating differential agronomic practices
for wheat and barley. (b) Relationship between ∆13C of
legumes (faba bean and lentil) and cereals (wheat and barley
combined), indicating the flat (r2=0.00) regression line
obtained (i.e. no relationship).
Published in Vegetation History and Archaeobotany 14:00-00 (2005) 107
preservation of different types of plant remains
(Wilson 1984; Wright 2003), the relative
frequencies of cereals and legume crops might
indicate that the area under grain legumes would
be very small compared with that of cereals. If this
were the case, such small, cultivated areas would
be probably devoted to labour-intensive crops (i.e.
garden crops) with supplementary irrigation, thus
explaining the high ∆13C values observed.
Intensive (i.e. under irrigation) cultivation of faba
bean could ensure a basic protein source in the
human diet (Heiser 1990). The combination of
extensive cereal crops expanding around human
settlements with smaller plots of vegetables and
legumes close to the living area has been reported
to be the most common pattern in subsistence and
pre-industrial agriculture (Hillman 1973). The
above data suggest that this kind of management of
water and soil resources could have been initiated
during the early phases of agriculture.
Further analysis of the relationship between
barley and wheat ∆13C across the sites (Fig. 4.4a)
can provide additional information. Despite both
species followed the same relationship between
water inputs and ∆13C (see Fig. 4.1), ∆13C values of
wheat in archaeological sites were generally lower
than those of barley (most points fell bellow the
1:1 reference line plotted in Fig. 4.4a). This
suggests that barley grains were grown with
greater water availability than wheat. Would this
mean that barley was selectively grown under
better conditions than wheat? Apparently not: if we
consider the current growth cycles of the two
species, we will find that barley grows up faster
than wheat, reaching maturity about two weeks
earlier. Thus, even growing at the same site, barley
grains in Mediterranean climates are generally
formed under moister conditions than wheat
grains, as drought has a steep increase during the
last weeks of the crop cycle (May-June).
Consequently, it is expected barley grains show
generally higher ∆13C values than the co-occurring
wheat grains. However, in Fig. 4.4a there are some
points where wheat has a considerably greater ∆13C
than barley, falling far away from the fitted
regression line (St. Vicenç Enclar, Sitges UAB and
Montou, in NE Spain; Puente Tablas and Peñalosa,
in SE Spain, see also Table 4.1). It is likely that
these potential outliers indicate some selective
treatments to enhance the performance of wheat
crops, but not applied in barley, including
irrigation or sowing in naturally wet alluvial soils
(Araus et al. 1997b; Bar-Yosef et al. 1989). When
we plotted in Fig. 4.4a present-day values of
rainfed barley, compared with either rainfed or
irrigated wheat, we found that the plots for rainfed
wheat fitted well within the regression line for the
archaeological sites. In contrast, the plots of
rainfed barley related to irrigated wheat fell out of
the 95% confidence prediction intervals of the
regression line, and showed values for wheat ∆13C
similar to those of the presumed "archaeological“
outliers. This further supports the idea that sites
showing greater wheat ∆13C involved a differential
water management for wheat and barley. As barley
is less drought-sensitive than wheat, it is a
common practice in dry areas to keep barley as a
rainfed crop, reserving any additional water
supply, or the moister soils (e.g. closer to a water
stream) for wheat. Currently, this practice is
enhanced by the fact that most barley is used for
animal feeding, whereas wheat is preferred for
human consumption. Again, we can find evidences
of water and soil management practices during
early agriculture that resemble those currently
found in some areas. This could be of interest not
only to reconstruct ancient agronomic techniques,
but also to evaluate the potential long-term impact
of current practices, looking at their consequences
in the past.
108
 Chapter 4. Water management practices and climate in ancient agriculture
4.4. Conclusions
Despite the drawbacks of inferring past
conditions based on present relationships, this
novel approach can help to identify events in
which early farmers started to develop a conscious
water management to improve their crops
performance. It seems also possible to relate these
events to the climatic conditions in which they
were developed, or look for possible changes in
response
to
climate
and/or
landscape
modifications. In particular, our results suggest
that, in general, ancient cereals (both wheat and
barley) were grown under better water status than
present-time crops. The more favourable water
conditions were probably due to climatic
variations, as suggested by the relatively good
agreement between crop and forest plant remains.
Nevertheless, we found evidences that, in some
cases, wheat was probably favoured by ancient
farmers, sowing it in better soils than barley, or
under supplementary irrigation. Our results also
indicate that grain legumes were usually grown
under more humid conditions than cereals, most
likely in small irrigated plots.
Acknowledgements
This work was partly supported by the CICYT project
BTE2001-3421-C02 and the EC project MENMED
(INCO-MED-ICA3-CT-2002-10022). We thank the
useful comments from F. Bittmann, S. Jacomet, M.
Charles and J.L. Vernet, which have contributed
significantly to improve the original manuscript. J.P.
Ferrio has a PhD fellowship from the Generalitat de
Catalunya.
Published in Vegetation History and Archaeobotany 14:00-00 (2005) 109
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Chapter 5
“…y aunque en rigor no es mejor,
por ser mayor o menor,
una encuesta hice a mi alrededor.”
Javier Krahe. Un burdo rumor
113
Chapter 5
J.P. FERRIO1 • N.ALONSO2 • J.VOLTAS1 • J.L. ARAUS3*
Estimating grain weight in archaeological cereal crops: a quantitative
approach for comparison with current conditions
1
Departament de Producció Vegetal i Ciència Forestal, E.T.S.E.A - Universitat de Lleida,
Av. Rovira Roure 191, Lleida E-25198, Spain
2
Departament d’Història, Universitat de Lleida, Victor Siurana 1, Lleida E-25003, Spain
3
Unitat de Fisiologia Vegetal, Facultat de Biologia, Universitat de Barcelona,
Diagonal 645, Barcelona E-08028, Spain
*Corresponding author; e-mail: [email protected]
Abstract Data relating to grain weight of cereal crops cultivated in the past could be useful to track
early genetic and/or agronomic improvements, facilitating the comparison of archaeological data with
current agronomic studies. However, archaeological grains are usually preserved by a process of
carbonisation, during which there is a considerable reduction in mass and dimension changes. The aim
of this study was to develop a model for the estimation of original grain weight from the dimensions
of charred grains, taking into account the effect of carbonisation. For this purpose, extant grains of
wheat and barley were experimentally carbonised at three temperature levels (200ºC, 250ºC, 300ºC)
and under two atmospheric conditions (oxidant, reducing). Weight, length (L), breadth (B) and
thickness (T) were measured before and after carbonisation. The products L×B and L×T provided the
best estimation of grain weight (r2=0.80-0.86) and were relatively stable across treatments. As a case
study, we applied grain weight models to wheat and barley grains gathered from a range of
archaeological sites in the Segre and Cinca Valley (Catalonia, NE Spain, ca. 3900-2200 cal. BP). In
this region, we observed a relatively constant increment in grain weight during the first half of I
millennium B.C.E.; probably explained by the increased water availability indicated by
palaeoenvironmental studies. In contrast, the increased grain weight of current samples, compared
with that of archaeological grains, is largely attributable to more recent genetic improvements.
Keywords agriculture • charred seeds • dimensions • morphology • carbonisation • wheat • barley
5.1. Introduction
Grain weight is one of the three main
agronomic components of grain yield in cereals,
along with the number of spikes per unit grown
area and the number of grains per spike; moreover,
it has direct implications for grain quality (MolinaCano et al. 1997; Evers et al. 1995). Therefore,
data relating to grain weight of cereal crops
cultivated in the past would be of interest as a way
to track genetic improvement in this trait, but also
as an indicator of the potential quality of the food
products that could be delivered from them.
Although strongly genetically determined, grain
weight also depends on environmental constraints,
such as water availability or temperature stress
(Voltas et al. 1999b; Rharrabti et al. 2003). For
example, when crops are initially grown under
favourable conditions, but become stressed during
Published in Journal of Archaeological Science 31:1635-1642 (2004) 115
grain filling (which is a common case in rainfed
Mediterranean environments), the time span for
grain growth is reduced and grain weight is limited
(Gooding et al. 2003; Savin and Molina-Cano
2002). Thus, estimates of grain weight in ancient
cereal crops could also provide helpful information
about the agronomic conditions under which the
plants were grown.
From an archaeological perspective, current
excavation techniques often involve the systematic
recovery of carbonised plant remains, including
grains from cereals and other crops, and grain
dimensions are routinely measured. However,
grain proportions change considerably after
charring and, consequently, are not directly
comparable with those of intact grains (Hopf 1955;
Lone et al. 1993). Similarly, although
archaeological grains can be weighed, their weight
is not directly comparable with the original weight,
as there can be considerable loss during
carbonisation (Threadgold and Brown 2003).
Moreover, grains are often found partially broken
and/or with mineral inclusions, still allowing
reliable measurements of principal dimensions, but
not of the total weight. In contrast, current
agronomic studies are conducted with noncarbonised grains and, whereas grain dimensions
are rarely considered, grain weight is measured
routinely (Cascón 1934; Henry and Kettlewell
1996;
Savin
and
Molina-Cano
2002).
Consequently, being able to estimate grain weight
in archaeological grains would allow us to
compare them with the large body of modern
agronomic data currently available. Hence, the aim
of this work was to find a way to link
archaeobotanic and agronomic studies, as the
comparison between such extensive sources of data
might help to answer some unsolved questions.
Our specific objective was to develop a model to
estimate original grain weight from charred grain
dimensions that would be applicable to
archaeological cereal seeds. We analysed the effect
of carbonisation on principal grain dimensions
(length, breadth and thickness), morphological
indices and grain weight. A wide range of
carbonisation levels, from slightly toasted to fully
carbonised or nearly combusted, under either
aerobic or anaerobic conditions, was assayed. By
including such a range of charring conditions in
model calibration, we expected to cover the
variability found in archaeological assemblages. A
case study on archaeological seeds recovered from
several sites in the Segre and Cinca Valley
(Catalonia, NE Spain) is presented to illustrate the
application of these models in an archaeological
context.
5.2. Experimental procedure
5.2.1. Plant material and charring conditions
The relationship between charred grain
dimensions and grain weight was modelled using
extant grains of barley (Hordeum vulgare L.) and
wheat (Triticum aestivum L.), harvested in the
experimental fields of UdL-IRTA centre in Lleida
(Catalonia, NE Spain). We took 144 grains for
each species, covering a wide range of grain
weight, dimension and morphology (Table 5.1).
We assigned grains to six separate subsets,
selected to include a similar range of grain weight
and dimensions. We ensured that there were no
significant differences in grain weight and
dimensions among the subsets. All samples were
dried at 60ºC for 24 hours, and submitted to a preheating ramp of two hours to minimise grain
explosion (i.e. ‘pop-corn’ effect) due to violent
output of water and combustible gases. The subsets
were carbonised under three levels of maximum
temperature (200ºC, 250ºC, 300ºC) and two
atmospheric conditions (oxidant, reducing).
Samples were kept at these temperatures for 45
min. To obtain a reducing atmosphere, three of the
subsets were buried in high crucibles under 3 cm
of sand, whereas the other three (oxidant) were
placed in flat crucibles without burying.
5.2.2. Grain measurements
Weight and dimensions were determined before
and after the experimental carbonisation of extant
grains. We measured length (L), breadth (B) and
thickness (T) under a stereoscopic microscope with
an LCD camera coupled to a PC, assisted by image
analysis software (Leika Qwin Lite 2.1, 1997).
After carbonisation, some grains were too distorted
and/or broken to be measured and were not
included in the analysis.
116
 Chapter 5. Estimating grain weight in archaeological cereal crops: a quantitative approach
Table 5.1 Mean and range for grain weight, dimensions and
morphological indices before and after experimental
carbonisation. The data shown here correspond to global
values, i.e. across the six treatments assayed. N, number of
samples.
Grain
Variable
Intact grains
Range Mean
Wheat
Grain weight (mg)
Length (L, mm)
Breadth (B, mm)
Thickness (T, mm)
L/B
L/T
B/T
Barley
Grain weight (mg)
Length (L, mm)
Breadth (B, mm)
Thickness (T, mm)
L/B
L/T
B/T
N=144
7.3 - 61.4
4.1 - 7.4
1.5 - 3.6
1.4 - 3.4
1.8 - 3.7
1.9 - 4.5
0.7 - 1.7
N=144
3.4 - 56.5
3.8 - 7.7
1.6 - 3.7
0.7 - 3.0
1.7 - 3.0
2.2 - 7.4
1.2 - 2.5
29.5
5.7
2.6
2.4
2.2
2.4
1.1
28.0
6.3
2.9
2.1
2.2
3.1
1.4
Charred grains
Range Mean
N=95
3.5 - 55.0
3.4 - 7.1
1.9 - 5.4
1.6 - 4.8
0.9 - 2.8
1.0 - 3.4
1.0 - 1.5
N=92
3.4 – 48.5
3.8 - 7.9
1.9 - 4.7
1.1 - 4.1
1.2 - 2.7
1.6 - 4.8
1.0 - 2.1
20.9
5.1
3.3
2.7
1.5
1.8
1.2
20.0
6.1
3.3
2.6
1.9
2.5
1.3
5.2.3. Statistical analyses
Data were subjected to analysis of variance
(ANOVA) to determine the effect of carbonisation
treatments
(temperature
and
atmospheric
conditions). Linear regressions were calculated to
assess the relationships between original grain
weight and dimension variables. Unless otherwise
stated, differences were considered statistically
significant when P<0.05. All analyses were carried
out using standard SAS-STAT procedures (SAS
1988).
Model fitness was assessed using root mean
square error (RMSE):
Eq. 5.2
(X − Xm)
1
+
2
n
∑X
2
SE p = RMSE *
where RMSE and n are as described above, X is
the value of the independent variable for a given
sample and Xm stands for the mean value of X over
all samples.
Grain weight of archaeological seeds was
estimated from the products Length x Breadth
(L×B) and Length x Thickness (L×T), taking the
mean value of both estimates. RMSE could not be
calculated for archaeological samples, as Yref (i.e.
real values) were unknown. Instead, we took root
mean square difference (RMSD) between the
values estimated from the two variables (L×B and
L×T), as a rough indicator of the prediction error in
archaeological samples:
Eq. 5.3
RMSD =
∑(Y
LB
n
where YLB and YLT stand for the predicted values
using either the L×B or L×T model, and n is the
number of samples. RMSD was also calculated
within calibration samples as a reference value for
archaeological results.
The overall standard error for a given
archaeological sample (SEsample) and site (SEsite)
was calculated following the general rules of error
propagation (Ku 1966):
Eq. 5.4
SEsample =
( SE pLB )2 + ( SE pLT )2
2
Eq. 5.1
RMSE =
∑ (Y
ref
− YLT )2
+ ( SELB , LT )2
Eq. 5.5
− Y pred ) 2
(n − p)
SE site =
where Yref and Ypred are observed and predicted
values for each sample, n stands for the number of
samples and p is the number of estimated
parameters (p=2). From this value we could obtain
the standard error (SEp) for each predicted value,
which is a function of all errors associated with the
regression equation (Bowley 1999):
∑ ( SE
sample
)
2
N
where SEpLB and SEpLT are the SEp associated
with the estimation of grain weight from L×B and
L×T, respectively, SELB , LT represents the standard
error of the mean between the two estimates (from
L×B and L×T) and N stands for the number of
samples within each site.
Published in Journal of Archaeological Science 31:1635-1642 (2004) 117
5.3. Results and discussion
5.3.1. Changes in grain size and morphology
during carbonisation
Overall, both grain weight and grain
dimensions changed considerably after charring,
thus confirming that they are not directly
comparable with those of intact seeds (Table 5.1).
We observed an average depletion in grain weight
of about 30%, and the grains became more
rounded, especially in wheat. There was a general
decrease in L (11% in wheat, 5% in barley),
whereas B and T increased (for B: 27% in wheat,
14% in barley; for T: 13% in wheat and 23% in
barley). Similar changes in grain dimensions
during carbonisation have been reported previously
for several species of wheat and barley, under a
variety of carbonisation conditions (Hopf 1955;
Lone et al. 1993; Willcox 2004; Renfrew 1973;
Colledge 1994). In all cases, charred grains tended
to shrink in length, whilst increasing in breadth and
thickness. In spite of these general trends, the
quantitative effect of charring varied considerably
according to the species and experimental
conditions. This is partly confirmed by our work,
as grain weight and dimensions after charring
differed significantly between temperatures (Table
5.2). However, we did not find any significant
effect of atmospheric conditions on the variables
studied.
Comparing the two species, we observed that
the degree of distortion was somewhat greater in
wheat than in barley. Probably, the greater density
(i.e. tighter packing) of wheat (75-80kg/hl) than
Table 5.2 Effect of carbonisation conditions on weight and grain dimensions, and determination coefficients (r2) of the linear
relationship (across treatments) between initial grain weight and different variables measured after carbonisation. Mean values
for intact grains (i.e. before carbonisation) are included for reference (N=144). Significance of the effect of temperature,
atmosphere and their interaction is indicated for each variable (* p<0.05, ** p<0.01, *** p<0.001). Mean values with the same
letter are non-significantly different according to the LSD test. Linear regressions are all significant at p<0.001. Measurable
samples (%), percentage of the original samples still measurable after carbonisation; L, length; B, breadth; T, thickness.
Treatment
Measurable Grain
samples (%) weight (mg)
Intact grains
Wheat
Barley
Charred grains
Wheat
Temperature
Atmosphere
Temp. × Atm.
Mean values
200ºC
100
250ºC
69
300ºC
29
r2 (across treatments)
Barley
Temperature
Atmosphere
Temp. × Atm.
Mean values
200ºC
100
250ºC
73
300ºC
19
2
r (across treatments)
29.5
28.0
***
n.s.
n.s.
27.7 a
15.4 ab
10.5 ab
0.67
***
n.s.
n.s.
25.3 a
14.8 b
12.2 b
0.69
L
(mm)
B
(mm)
T
(mm)
L*B*T
(mm3)
B*T
(mm2)
L*B
(mm2)
L*T
(mm2)
5.7
6.3
2.6
2.9
2.4
2.1
36
38
06.2
06.1
14.8
18.3
13.7
13.2
***
n.s.
n.s.
n.s.
n.s.
n.s.
*
n.s.
n.s.
n.s.
n.s.
n.s.
*
n.s.
n.s.
5.5 a
4.6 b
4.6 b
0.43
3.3 a
3.4 a
3.5 a
0.50
2.6 a
2.9 ab
2.9 b
0.49
48 a
47 a
50 a
0.77
08.6 a
10.0 ab
10.5 b
0.52
*
n.s.
n.s.
**
n.s.
n.s.
***
n.s.
n.s.
**
n.s.
n.s.
***
n.s.
n.s.
6.3 a
5.8 b
6.3 ab
0.64
3.2 a
3.4 a
3.8 b
0.67
2.4 a
2.8 b
3.3 c
0.61
51 a
56 ab
85 b
0.77
07.8 a
09.5 b
12.8 c
0.65
118
 Chapter 5. Estimating grain weight in archaeological cereal crops: a quantitative approach
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
18.2 a
16.0 a
16.0 a
0.80
14.6 a
13.4 a
13.7 a
0.82
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
20.5 a
19.8 a
24.7 a
0.84
15.2 a
15.9 a
21.0 a
0.82
barley grains (60-65kg/hl) enhanced the distortion
during charring. Nevertheless, despite showing
greater deformations, they were less dependent on
the carbonisation environment for wheat than for
barley (Table 5.2). In wheat, L was the most
variable dimension, whereas for barley T was the
least consistent. As our aim was to develop a
model sufficiently robust to be applied over a wide
range of seed morphologies, the best variables
would be those that are relatively stable regardless
of carbonisation conditions. The products L×B and
L×T were the only two variables not showing
significant differences among treatments in both
species. Apparently, the increment in either B or T
brought about by higher temperatures was
compensated by the reduction in L, thus giving a
more stable variable than each dimension taken
separately. In addition, L×B and L×T showed the
strongest relationships with grain weight across all
treatments (Table 5.2). Consequently, the variables
of choice for model calibration were L×B and
L×T. As expected, although the weight of charred
grains showed a relatively good correlation with
original grain weight across treatments, it was
highly dependent on the charring conditions.
Indeed, grain weight showed little variation when
carbonisation was performed at 200ºC (the lowest
temperature assayed), but was reduced drastically
(about 50%) at temperatures above 250ºC (Table
5.2). This, added to the aforementioned
considerations (grain fragmentation, presence of
mineral inclusions and other taphonomic
alterations), confirms that this variable cannot be
used as an estimator of original weight in
archaeological cereal seeds.
5.3.2. Sample preservation: does initial grain
weight affect sample survival during charring?
As the degree of carbonisation increased, a
greater number of samples were either destroyed or
too distorted to be measured (Table 5.2). In order
to account for potential drifts due to sample loss,
we checked whether or not the “surviving” seeds
differed in their original grain weight when
compared with the rest of the samples. There were
no significant differences (data not shown) and,
consequently, no evidence of a differential
preservation of seeds related to their initial grain
weight. Moreover, we did not find any significant
relationship between original grain weight and the
changes in grain dimensions during carbonisation.
This suggests that grain size of a carbonised cereal
sample is likely to be representative of the original
sample, provided that there is no further drift
during burial and recovery. Wilson (1984) and,
more recently, Wright (2003), assayed the effect of
different carbonisation environments on the
relative survivability of weed seeds, both
concluding that it was strongly dependent on the
heating conditions. In a series of field experiments,
including experimental fires, sampling and
flotation, Gustafsson (2000) also found that weed
seeds showed contrasting levels of carbonisation,
even under the same conditions, and suggested that
this could be related to substantial differences in
seed composition (e.g. oil content). However, in
the same experiment, grains from different cereal
species were carbonised and recovered in similar
proportions, probably due to their relatively
uniform chemical composition. Considering the
great morphological variability found among
cereal species, the work of Gustafsson supports the
absence of any differential preservation of cereal
grains according to their size and/or morphology.
Nevertheless, random drifts should not be
discounted, although they can be minimised by
increasing sample size, as well as including
samples from several carbonisation events
(Gustafsson 2000; Boardman and Jones 1990;
Buxó 1997).
5.3.3. Grain weight models:
performance and range of application
Once the most suitable variables were selected,
we used regression models to predict grain weight,
pooling samples from all treatments together, with
the aim of covering the widest range of
experimental situations. In both species, L×B and
Published in Journal of Archaeological Science 31:1635-1642 (2004) 119
L×T were strongly related to grain size (r2=0.800.86), although they showed different trends in
wheat and barley (Fig. 5.1). In wheat, both L×B
and L×T were linearly related to grain size.
However, in barley the relationship between these
variables and grain size was improved using a
power model (Fig. 5.1c, d). This could be
explained by the lower morphological distortion of
barley grains, as the relationship between these
variables and weight in intact grains of both
species fitted power curves (data not shown).
RMSE values of 5.1-5.4 mg (Fig. 5.1) suggest a
good predictive accuracy of the models.
Nevertheless, taking the mean between the values
estimated from L×B and L×T gave the most
reliable estimations of grain weight, and RMSE
was lower (4.9 mg and 5.0 mg, for wheat and
barley, respectively) than for each of them taken
separately. Within the whole calibration range,
SEp was always less than 2 mg, and it was less
than 1 mg in the most common ranges of 9-44 mg
and 10-46 mg of predicted values, for wheat and
barley, respectively. It should be noted that the
range of dimensions and morphology included in
calibration was similar to that usually reported in
archaeological grains for both species, which can
be 3.0-7.5 mm in L, 1.7-4.6 mm in B and 1.5-4.0
mm in T (Willcox 2004; Colledge 2001; Buxó
1997; Lone et al. 1993; Alonso 1999; Alonso
1986; Alonso et al. 2002). Moreover, mean site
60
Grain weight (mg)
a) Wheat
b) Wheat
50
40
30
20
Y=-15.1+2.98x
2
N=95 r =0.82***
RMSE=5.1mg
Y=-15.4+2.47x
2
N=95 r =0.80***
RMSE=5.3mg
10
0
Grain weight (mg)
c) Barley
d) Barley
50
40
30
20
1.28
1.60
Y=0.78x
2
N=92 r =0.85***
RMSE=5.4mg
Y=0.21x
2
N=92 r =0.86***
RMSE=5.1mg
10
0
5
10 15 20 25 30 35
L*B (mm2)
0
5
10 15 20 25 30 35
L*T (mm2)
Fig. 5.1 Regression models across treatments to estimate initial grain weight of wheat (a, b) and barley (c, d) from the products
Length x Breadth (L×B; a, c) and Length x Thickness (L×T; b, d) of carbonised grains. Circles, triangles and squares indicate
temperature levels of 200ºC, 250ºC and 300ºC, respectively. Open and closed symbols indicate reduction and oxidation
treatments, respectively. All regressions were significant at p<0.001.
120
 Chapter 5. Estimating grain weight in archaeological cereal crops: a quantitative approach
values reported in the literature would always be
included within the aforementioned optimal range
(SEp<1 mg). This suggests that current models can
be applied to most archaeological cereal seeds,
provided that they are sufficiently well preserved
to allow reliable measurements.
5.4. A case study with archaeological
samples
To further assess the suitability of the models,
we applied them to a set of archaeological samples.
Charred grains of naked wheat (Triticum
aestivum/durum) and hulled barley (Hordeum
vulgare) were gathered from six archaeological
sites in the Segre and Cinca Valley (Catalonia, NE
Spain, see Table 5.3), ranging from Bronze Age
(ca. 3900 cal. BP) to Second Iron Age (Late
Iberian period: ca. 2200 cal. BP). Currently, all
these sites show similar climatic and soil
conditions, and are characterised by a semiarid
Mediterranean climate. The samples were gathered
from various archaeological contexts, such as
domestic fires, cooking ovens, storage jars, room
floors and levels of rubble from housing structures
and pits (for further details about the sites and
sampling procedures, see Alonso 1999; Alonso
1986; Alonso et al. 2002). We determined grain
dimensions in these samples as described in
section 5.2.2. Seeds too distorted and/or broken
were not measured.
Firstly, we confirmed that the range of values
within the set of archaeological samples was
suitable for the application of the models. Thus,
96% of the samples fell within the calibration
range for grain dimensions and morphological
indices described in Table 5.1, with about 95% of
the samples falling within the optimal range
(SEp<1 mg) of the models. Removing the samples
that fell outside of these limits did not significantly
affect the results, so we decided to keep them in
the analysis. Moreover, calculated RMSD for
archaeological samples (3.9 mg and 3.4 mg, for
wheat and barley, respectively) was similar to that
found within calibration samples (3.6 mg for both
Table 5.3 Site description and number of archaeological grains
used in the case study. Calibrated age represents the
approximate age in years BP estimated from a combination of
archaeological and calibrated 14C dating using the program
CALIB 3. 03 (Stuiver and Reimer 1993).
Site
Lat.
Samples
Long. Cultural Calibrated
Period
Age (BP) Wheat Barley
Minferri 41º30’ 0º45’ Bronze
Age
El Vilot 41º38’ 0º30’ Bronze
III
Age
El Vilot 41º38’ 0º30’ Bronze
Age
II
Els
41º34’ 0º56’ First
Vilars II
Iron Age
Margalef 41º34’ 0º47’ Second
Iron Age
Tossal 41º37’ 0º47’ Second
Tenalles
Iron Age
4100-3650
118
029
3113-2794
013
017
2923-2393
014
070
2550-2425
092
170
2250-2150
100
100
2250-2150
100
100
species), suggesting that the accuracy of the
estimates in archaeological seeds was close to
those reported for current samples (section 5.3.3).
Fig. 3.2 plots average grain weight for the six
archaeological sites, taking the mean between the
estimates from L´B and L´T models, as described
in section 5.2.3. We also included a reference
value for present-day samples of wheat (Araus et
al. 2003; A.E.T.C. 2003) and barley (Voltas et al.
1997; Voltas et al. 1999a) grown in rain-fed
conditions in the same area. We observed a large
increase in grain weight of present-day samples as
compared with that of archaeological grains.
Indeed, even under the harsher conditions in which
barley and wheat can grow, values of grain weight
are usually over 30 mg, and rarely fall below 25
mg (Gooding et al. 2003; Acevedo et al. 1991;
Araus et al. 1998). Such results contrast with the
estimated grain size among the archaeological sites
studied, which were always below 25 mg.
Moreover, when we applied our models to other
archaeological data acquired from the literature
(Buxó 1997; Lone et al. 1993), estimated grain
weight was never greater than 30 mg. Data from
Published in Journal of Archaeological Science 31:1635-1642 (2004) 121
geomorphology (Gutiérrez and Peña 1992),
antracology (Ros 1997; Alonso et al. 2002) and
palynology (Alonso et al. 2002) indicate that the
current climate in the area is more arid than in the
period studied. In addition, the analysis of carbon
isotope composition of barley and wheat grains
recovered from other archaeological sites in
Catalonia also suggested that they were grown
under much wetter conditions between the
Neolithic period and the Middle Ages than
currently (Araus and Buxó 1993). Thus, such a
large increase in grain weight from archaeological
to present times is mostly attributable to more
recent advances in plant breeding and agronomic
practices, including the extensive application of
nitrogen fertilisers (Rharrabti et al. 2003; Acevedo
et al. 1991; Gooding et al. 2003; Voltas et al.
1998). The differences observed among
archaeological samples, however, appear to be
related to environmental changes in the studied
area. At the second half of the II millennium
B.C.E., vegetation was characterised by evergreen,
schlerophyllous species, forming associations of
Rhamno-Quercetum cocciferae and RosmarinoEricion, typically Mediterranean (Ros 1997;
Alonso et al. 2002). During the first half of the I
millennium B.C.E., however, there was a
progressive change in vegetation, with the
formation of meso/submediterranean forests,
composed of a mixture of oaks (evergreen and
deciduous) and pines, together with shrub
woodlands in the driest places (Ros 1997; Alonso
et al. 2002). This is in agreement with the
existence of a period of wetter climate, according
to geomorphological studies (Gutiérrez and Peña
1992). Consequently, the increase in grain weight
observed from 3000 BP to 2000 BP, particularly
apparent in barley, may be due to changes in water
availability. Nevertheless, we cannot rule out the
possibility of genetic improvement in grain size
(possibly unintentional in agronomic terms) during
the period studied, as increased grain size implies
40
Grain weight (mg)
a) Wheat
b) Barley
35
30
25
20
15
10
4.0 3.5 3.0 2.5 2.0
Ky BP
0.0 4.0 3.5 3.0 2.5 2.0
0.0
Ky BP
Fig. 5.2 Evolution of grain weight of barley and wheat in the Segre and Cinca Valley (Catalonia, NE Spain) from Bronze Age
(ca. 3900 cal. BP) to Second Iron Age (ca. 2200 cal. BP), compared with present-day samples (see Table 3 for a description of
the archaeological sites). Archaeological values were estimated from the products Length x Breadth (L×B) and Length x
Thickness (L×T), taking the mean value of both estimates. Present-day data correspond to barley (Voltas et al. 1997; Voltas et al.
1999) and wheat (A.E.T.C. 2003; Araus et al. 2003) grown under rain-fed conditions around the studied area. Horizontal error
bars indicate the range of calibrated age. Vertical error bars of the archaeological samples indicate the standard error of predicted
means, including within-site variability, regression error and the difference between predicted values using the two models (see
text for details). For present-day data, vertical error bars indicate the standard error of the mean across environments (N=6).
122
 Chapter 5. Estimating grain weight in archaeological cereal crops: a quantitative approach
better end products. Indeed, grain weight is
directly associated with flour yield (Henry and
Kettlewell 1996) and baking properties of wheat
(Evers et al. 1995), and also determines malting
quality of barley (Molina-Cano et al. 1997).
Moreover, the differences between archaeological
and current data were not limited to modern
cultivars, but also applicable to traditional
landraces (Acevedo et al. 1991; Araus et al. 1998;
Cascón 1934), supporting the idea that genetic
improvement in grain size had already started
before the implementation of modern breeding
programs. Indeed, grain size is an easily
identifiable trait, and, for example, it has been
documented
that
Spanish
farmers
have
traditionally reserved the best seeds (i.e. the
bigger) for sowing (Cascón 1934): it is probable
that such practice began during the early phases of
agriculture.
Nevertheless, we should bear in mind that
carbonised plant remains are not necessarily a
random sub-sample of the overall harvest. Indeed,
carbonised grains might have been those discarded
by the farmers, and thrown onto fires along with
other plant debris (Dennell 1976; Boardman and
Jones 1990). Exceptionally, for two of the sites
studied (Margalef and Tossal de les Tenalles),
there was evidence that the samples analysed were
representative of harvested grains, as thousands of
them were found within storage jars. For the
remaining sites, however, we are unaware of the
origin of the grains and, although a large number
of samples (and carbonisation events) would
reduce any biasing, these results should be
interpreted with caution. Nevertheless, the
difference in grain weight between stored grains
and those recovered from other sources was much
smaller than that between archaeological and
present-day grains. Therefore, even considering
only
stored
material
(i.e.
undoubtedly
representative of harvested grains), we can
conclude that a significant genetic improvement in
grain weight occurred in the area, at least during
the last two millennia.
5.5. Conclusions
We have confirmed that although there is
considerable change in dimensions and
morphology of wheat and barley grains during
carbonisation, some measurements are relatively
constant over a wide range of carbonisation levels.
The products L×B and L×T were the best
correlated with grain weight, and also the most
stable across carbonisation treatments. The range
of dimensions and morphology present in the
sample set used for calibrations suggested that it
was representative of the variety of (unknown)
conditions in which archaeological grains were
carbonised. Indeed, this range was wide enough to
allow accurate predictions in about 95% of the
archaeological seeds in which the models were
tested. In our case study, we concluded that the
increase in grain weight between archaeological
samples and present-day crops was principally due
to genetic improvements. Differences among
archaeological samples, however, were apparently
related to environmental change; although genetic
differences could not be dismissed. Further work
will be necessary to clarify the origin of such
variations, as well as to determine the timing of the
change in grain weight to its current values.
Acknowledgements
This work was partly supported by the CICYT grant
BTE2001-3421-C02-01 and the INCO-MED project
MENMED (ICA3-CT-2002-10022). J.P. Ferrio has a
PhD Fellowship from Generalitat de Catalunya. We
thank the extensive crops department from UdL-IRTA
for providing extant seeds. We also acknowledge the
useful comments of the referees, which helped us to
significantly improve the original manuscript.
Published in Journal of Archaeological Science 31:1635-1642 (2004) 123
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Chapter 6
“Supongamos ahora que sembráis trigo candeal [...] el trigo crecerá de una
manera uniforme [...] si existe alguna diferencia entre las espigas, se deberá
a que habrán recibido distintos cuidados del labrador o a que habrán
participado desigualmente de los beneficios de la lluvia y el rocío [...]”
Kung Tsé. Meng Tsé
127
Chapter 6
J.P. FERRIO1* • N.ALONSO2 • J.VOLTAS1 • J.L. ARAUS3
Evolution of grain weight in archaeological cereal seeds: potential
role of climate and genetic improvement
1
Departament de Producció Vegetal i Ciència Forestal, E.T.S.E.A - Universitat de Lleida,
Av. Rovira Roure 191, Lleida E-25198, Spain
2
Departament d’Història, Universitat de Lleida, Victor Siurana 1, Lleida E-25003, Spain
3
Unitat de Fisiologia Vegetal, Facultat de Biologia, Universitat de Barcelona,
Diagonal 645, Barcelona E-08028, Spain
*Corresponding author; e-mail: [email protected]
Abstract Inferences on grain weight (GW) of cereal crops cultivated in the past could be useful to
track early agronomic improvements derived from empirical breeding. Though strongly genetically
determined, GW also depends on environmental constraints, such as water availability during grain
growth. In the archaeological context, it is possible to estimate GW from grain dimensions, while
plant water status can be determined by the study of carbon isotope discrimination (∆13C). The aim of
this work was to determine the relative effect of environmental changes on the evolution of GW of
naked wheat (Triticum aestivum/durum) and hulled barley (Hordeum vulgare) in the Segre and Cinca
Valley (Ebro Depression, NE Spain) during the last four millennia. We performed grain
measurements and δ13C analyses in a set of charred grains, recovered from nine archaeological sites,
spanning from Bronze Age (ca. 1950 BCE) to Middle Age (XI c. CE). Estimated GW in
archaeological grains (19.6 mg in average for both species) was significantly smaller than the values
found in present rainfed cereals cultivated in the area (32.5 mg). In contrast, average ∆13C values did
not differ significantly (15.93‰ and 15.90‰, for archaeological and modern material, respectively.
Accordingly, the most recent increase in GW cannot be attributed to increased water availability. We
concluded that the greater grain weight in present-day cereals responded to a genetic improvement of
the crops, which might have started during the Middle Age.
Keywords agriculture • charred seeds • dimensions • wheat • barley • Carbon isotope discrimination
(∆13C) • water availability
6.1. Introduction
Wheat and barley are among the first cultivated
crops, and its cultivation has been successively
improved across millennia, through a combination
of genetic selection and advances in agronomic
techniques. Grain yield (i.e. total weight of grains
per unit of cultivated area) is currently the trait of
greatest interest for agronomist, and it was crucial
for the survival and development of early farming.
It is defined by three numerical components: the
number of spikes per unit grown area, the number
of grains per spike, and individual grain weight
(GW). From the current knowledge, however, it is
not possible to find direct evidences of the values
To be submitted to Journal of Cereal Science 129
attained in the past for the first two components, at
least until the beginning of writing. Even in this
case, existing documentary records are often
difficult to interpret, as they are not easily
comparable with current agronomic studies.
Charred plant remains (including grains of cereals
and other crops) are routinely recovered from
archaeological sites, and dimensions and other
morphometric traits of cereal grains are
systematically measured (Buxó 1997; Renfrew
1973; Van Zeist and Casparie 1984).
Unfortunately,
grain
proportions
change
considerably after charring and, consequently, are
not directly comparable with those of intact grains
(Hopf 1955; Lone et al. 1993). Ferrio et al. (2004a)
developed a method to estimate the original GW of
cereal remains from the dimensions of charred
grains, which was robust enough to override the
effect of carbonisation. Unlike grain dimensions,
GW is routinely determined by agronomists, and
thus its quantification in charred grains allows to
interpret archaeobotanical data under the light of
existing agronomic knowledge.
Despite being strongly genetically determined,
GW also depends on climatic constraints, such as
water availability or temperature stress (Calderini
et al. 1999b; Rharrabti et al. 2003; Voltas et al.
1999). Specifically, the main factors defining GW
are 1) the amount of assimilates (carbohydrates)
per grain during grain filling and 2) the duration of
the grain filling period. The amount of assimilates
per grain depends on plant conditions during the
whole growth cycle, but also on the number of
grains. If plants become stressed early in the
growth cycle, both the assimilates and the number
of grains are reduced and individual grain size may
remain unaffected (Henry and Kettlewell 1996).
However, when crops are initially grown in
favourable conditions, but become stressed during
grain filling (which is a common case in
Mediterranean environments), the time span for
grain growth is reduced and grain size is strongly
limited (Gooding et al. 2003; Savin and MolinaCano 2002). Thus, changes in grain size of
cultivated cereals in the past may be due to either
environmental or genetic factors, or a combination
of both.
Ferrio et al. (2004a) applied their models to
estimate GW over a set of archaeological seed
remains from the Segre and Cinca Valley
(Catalonia, NE Spain), and they found significantly
lower GW during the period studied (ca. 2000-200
BCE) than in present times. They attributed such
differences in GW mostly to genetic changes
attained since the end of the first millennia BCE,
based on several palaeoenvironmental evidences
indicating that climate during the studied period
was more humid than present. However, before
discarding the effect of climatic constraints on
grain size, it would be helpful to obtain more direct
evidences of the water status in which
archaeological grains were developed, as it often
limits GW (Gooding et al. 2003; Savin and
Molina-Cano 2002). For C3 species, δ13C in plant
tissues constitutes an integrated record of the ratio
of intercellular to atmospheric partial pressure of
CO2 (ci/ca) during the time the tissue was
synthesized. In winter cereals, this ratio is mainly
determined by stomatal conductance and hence
δ13C in grains represents a good indicator of water
status during grain filling. Accordingly, the
measurement of δ13C from charred grains found in
archaeological sites has been proposed as a method
to evaluate water inputs (due to rainfall and/or
irrigation practices) in ancient agriculture (Araus et
al. 1997; Araus et al. 1999).
The specific aim of this work was to determine
the relative effect of environmental changes on the
evolution of GW in the Segre and Cinca Valley
during the last four millennia. For this purpose, we
performed δ13C analyses in a set of charred grains,
recovered from the same archaeological sites in
which grain measurements were taken. In order to
refine the timing of the observed changes in GW
from the archaeological context to present-times,
we also applied the models described in Ferrio et
al. (2004a) to estimate GW to new sets of
archaeological material from the same area,
covering a wider temporal and spatial range.
130
 Chapter 6. Grain weight in archaeological cereal seeds: role of climate and genetic improvement
6.2. Materials and Methods
6.2.1. Archaeological sites and plant remains
Charred grains of naked wheat (Triticum
aestivum/durum) and hulled barley (Hordeum
vulgare) were recovered from nine archaeological
sites in the Segre and Cinca Valleys (Mid Ebro
Depression, NE Spain, see Fig. 6.1 and Table 6.1),
including the temporal range between the Bronze
Age (ca. 1950 BCE) and Middle Ages (XI c. CE).
These sites now display similar climatic and soil
conditions, and are characterized by a semi-arid
Mediterranean climate. The samples were collected
from various archaeological contexts, such as
domestic fires, cooking ovens, storage jars, room
floors and levels of rubble from housing structures
and pits (for further details about the sites and
sampling procedures, see Alonso 1992, 1999,
2005; Alonso et al. 2002; Sarró excavation team
2001). For the sites from the Bronze and First Iron
Age, radiocarbon ages were determined at Beta
Analytical Inc. (Miami, USA) and at the
Universitat de Barcelona (Barcelona, Spain), and
calibrated dating was calculated with the software
CALIB REV 4.3 (Stuiver et al. 1998). For the
remaining sites, a combination of stratigraphic and
archaeological dating was used. To recover plant
remains, soil samples were treated using a standard
flotation tank in the field with 5 mm, 2 mm and 0.5
mm sieves, and taxonomical determinations were
performed on the basis of the morphological
features of the grains.
6.2.2. Carbon isotope composition
and meteorological data
Because of the carbonate enrichment of
archaeological sediments, we soaked each grain
separately with HCl 6M for 24h at room
temperature to remove carbonate crusts (DeNiro
and Hastorf 1985). The samples were then rinsed
repeatedly with distilled water and oven-dried at
60ºC for 24 hours before milling to fine powder for
carbon isotope analyses. δ13C was determined by
elemental analysis and isotope ratio mass
spectrometry (EA/IRMS) at Isotope Services, Inc.,
Fig. 6.1 Geographical location of the archaeological sites
included in our study, indicating, in the upper section of the
map, the isohyets of mean annual precipitation, and in the
lower section, altitude a.s.l.
Los Alamos, NM, USA. To account for changes in
δ13C of atmospheric CO2 (δ13Cair) during the
Holocene, we calculated carbon isotope
discrimination in grains (∆13Cg) from δ13Cair and
grain δ13C (δ13Cg), as described by Farquhar et al.
(1982):
Eq. 6.1
∆13Cg =
δ 13Cair − δ 13Cg
δ 13Cg
(1 +
)
1000
δ13Cair was inferred by interpolating a range of
data from the literature, covering the temporal
sample range (Eyer et al. 2005; Francey et al.
1999; Indermühle et al. 1999; Leuenberger et al.
1992).
To be submitted to Journal of Cereal Science 131
6.2.3. Estimation of grain weight
Grain dimensions of wheat and barley grains
were determined in all the studied sites (up to one
hundred grains per species and stratigraphic unit),
but excluding samples too distorted and/or broken
to be measured. We measured length (L), breadth
(B) and thickness (T) under a stereoscopic
microscope with an LCD camera coupled to a PC,
assisted by image analysis software (Leika Qwin
Lite 2.1, 1997).
GW of archaeological seeds was estimated
from the products Length x Breadth (L×B) and
Length x Thickness (L×T), taking the mean value
of both estimates, as described in Ferrio et al.
(2004a):
For wheat:
Eq. 6.2
6.2.5. Statistical analyses
We fitted locally weighted least squares
regression curves (LOESS, Cleveland 1979) to the
data sets to objectively summarize overall trends in
the time series of the studied variables (GW, ∆13C).
Linear regressions between ∆13C and estimated
GW were built on a subset of samples, in both
variables were simultaneously available, to model
the effect of water status on grain size. Differences
in such relationship between wheat and barley
were determined using an heterogeneity of slopes
ANOVA. From such regressions, we calculated
residual GW values, to quantify the variability not
associated to water availability during grain filling.
To get a more robust plot of the evolution of GW
residuals, missing values of either ∆13C or GW
were filled using the estimations given by LOESS
curves.
GW = −15.4 + 2.47 ⋅ ( L × B )
Eq. 6.3
GW = −15.1 + 2.98 ⋅ ( L × T )
For barley:
Eq. 6.4
GW = 0.21× ( L × B)
1.60
Eq. 6.5
GW = 0.78 × ( L × B)1.28
6.2.4. Estimation of water inputs
during grain filling
Total water inputs (TWI) during grain filling
were estimated from the ∆13C of charred grains,
following the models defined in Araus et al. (1997;
1999), as modified by Ferrio et al. (2005a):
For wheat:
Eq. 6.6
TWI = 0.175 × e
(0.376×∆13C)
For barley:
Eq. 6.7
TWI = 0.225 × e
(0.364×∆13C)
132
 Chapter 6. Grain weight in archaeological cereal seeds: role of climate and genetic improvement
Table 6.1 Dating, cultural period, average grain dimensions and estimated grain weight for the archaeological sites (see Fig. 6.1).
N, number of grains measured; L × B × T, grain dimensions (length × breadth × thickness); GW, estimated grain weight.
Archaeological Calendar year
(BCE/CE)
site
1. Minferri
1946 cal. BCE
1875 cal. BCE
1673 cal. BCE
2. El Vilot de
1412 cal. BCE
Cultural
Period
Early
Bronze Age
Middle
Bronze Age
Montagut
888 cal. BCE Late
773 cal. BCE Bronze Age
773 cal. BCE
3. Masada del 1000 cal. BCE Late
Ratón
Bronze Age
4. Tozal de los
896 cal. BCE First
Regallos
750 cal. BCE Iron Age
5. Els Vilars
793 cal. BCE First
781 cal. BCE Iron Age
500 BCE
Second
Iron Age
6. Margalef
200 BCE
Second
Iron Age
7. Roques Sarró 200 BCE
Iron Age
8. Tossal de les 200 BCE
Second
Tenalles
Iron Age
9. Ciutat de
50 CE
Roman
Lleida
300 CE
1025 CE
Middle Age
1040 CE
Total
N
32
22
47
10
Wheat
L × B × T (mm)
4.6 × 3.1 × 2.7
4.5 × 2.8 × 2.4
4.4 × 2.9 × 2.6
4.4 × 3.2 × 2.7
5.0 × 2.9 × 2.1 15.9 ± 1.4
5.0 × 2.8 × 2.3 16.3 ± 1.9
5.5 × 2.9 × 2.3 19.0 ± 0.7
4.2 × 2.4 × 1.8 9.4
13
1
14
4
4.4 × 2.9 × 2.4
4.7 × 3.0 × 2.4
4.5 × 2.9 × 2.5
4.3 × 2.6 × 2.4
16.8 ± 1.7
19.1
17.9 ± 1.4
14.7 ± 2.9
4 4.9 × 2.7 × 2.1 14.3 ± 2.2
2 5.7 × 2.6 × 2.1 17.0 ± 4.3
170 5.6 × 2.9 × 2.3 19.8 ± 0.4
1
3
87
4.3 × 2.3 × 1.8
4.6 × 2.6 × 2.2
4.5 × 2.8 × 2.4
8.4
15.2 ± 3.4
16.8 ± 0.6
100 5.7 × 2.9 × 2.2 19.0 ± 0.3
100
5.0 × 3.0 × 2.6
22.4 ± 0.5
100 6.1 × 3.0 × 2.4 23.2 ± 0.5
6
100
4.5 × 2.6 × 2.2
5.1 × 2.9 × 2.5
14.1 ± 2.3
21.9 ± 0.4
2 5.9 × 3.0 × 2.3 20.7 ± 1.9
6 5.7 × 2.7 × 2.1 17.6 ± 1.5
506 5.6 × 2.9 × 2.3 19.6 ± 0.2
5
8
2
3
4.8 × 3.1 × 2.3
4.4 × 3.0 × 2.5
5.7 × 3.3 × 2.5
4.8 × 3.0 × 2.8
4.7 × 2.9 × 2.5
19.9 ± 2.0
17.5 ± 2.3
28.7 ± 1.9
22.6 ± 0.5
N
24
1
3
2
17
6
68
1
Barley
L × B × T (mm)
4.5 × 2.9 × 2.1
5.7 × 3.2 × 2.4
4.5 × 2.3 × 2.0
4.9 × 2.6 × 2.1
6.3. Results and Discussion
6.3.1 Grain weight and carbon isotope values
in archaeological material
Grain dimensions of charred seeds showed a
considerable
range
of
variation
across
archaeological sites (see Table 6.1), ranging from
9.4 to 23.2 mg in the case of barley, and from 8.4
to 28.7 mg for wheat. When using regression
models to estimate a given variable, reliable
estimations are only obtained for samples lying
within within the range of variation used for
calibration. In our case, over 95% of the grains
were well inside the ranges of L×B and L×T used
by Ferrio et al. (2004a) to calibrate GW models
GW (mg)
14.1 ± 0.9
21.3
10.9 ± 0.6
13.7 ± 1.0
458
GW (mg)
20.3 ± 0.9
17.0 ± 1.4
17.5 ± 0.8
20.2 ± 1.6
19.6 ± 0.3
(Eqs. 6.2-6.3), indicating that such models are
appropriate for the present data. Estimated GW in
archaeological grains (19.6 mg in average for both
species) was considerably smaller than the values
found in present rainfed cereals cultivated in the
area (30 mg ±1.5 for barley and 35 mg ± 2.0 for
wheat, according to Ferrio et al. (2004a).
Average δ13C in grains for the sites studied also
varied considerably, ranging from -23.4 to -21.1 ‰
in barley, and from -23.0 and -20.8 ‰ in wheat
(Table 6.2). Estimated δ13C in atmospheric CO2
suffered relatively small changes during the period
considered, and thus calculated ∆13C showed
nearly the same range of variability found for δ13C
To be submitted to Journal of Cereal Science 133
(from 14.8 to 17.4 ‰, and from 14.2 to 17.0, for
barley and wheat, respectively). On average, ∆13C
in barley grains was higher than in wheat (16.1 ‰
±0.2 and 15.7 ‰ ±0.2, respectively). The same
general trend has been reported in previous studies
on archaeological material collected around the
Mediterranean (Araus et al. 1997; Araus et al.
1999; Ferrio et al. 2005a). Such differences in
isotope signature can be attributed to the shorter
growing cycle of barley, which reaches maturity
about two weeks earlier than wheat. As
Mediterranean climates are characterised by a
steep decrease in water availability (and rising
temperatures) by the end of the cereals growing
cycle (late spring), plants with shorter cycle would
suffer less water stress during grain filling, which
is translated into lower ∆13C in grains. In both
cases, the observed range of ∆13C felt within the
calibration range of the models used to predict
TWI (Eq. 6.6-6.7, see Araus et al. 1997; 1999;
Ferrio et al. 2005a), implying that these models
can give reliable estimations of TWI for our
samples. Estimated values of TWI were highly
variable, but they are still within the range of
values reported for rainfed cereals cultivated
around the archaeological sites: between ca. 30150 mm for barley (Araus et al. 1997; Voltas et al.
1997) and between ca. 50-110 mm for wheat
(Araus et al. 2003; Ferrio et al. 2005b). Moreover,
average TWI in archaeological grains did not differ
significantly from the estimations derived from
present ∆13C values in grains (82 mm ±14 for
barley and 62 mm ±20 for wheat, after (Ferrio et
al. 2005a). It should be noted that, despite showing
differences in ∆13C, estimations of TWI for
archaeological wheat and barley did not differ
significantly. This means that, for a given ∆13C,
TWI in wheat would be greater than in barley.
Again, this would agree with the shorter cycle of
barley: even receiving the same amounts of water
during grain filling (i.e. TWI), wheat plants would
become more stressed during this period because
of a greater vapour pressure deficit, driven by
higher temperatures.
Table 6.2 Dating, estimated δ13C in atmospheric CO2 (δ13Cair), average carbon isotope composition (δ13C), discrimination (∆13C),
and estimated water inputs during grain filling (TWI) in charred grains recovered from each archaeological sites.
Archaeological Calendar year
site
(BCE/CE)
1. Minferri
1946 cal. BCE
1673 cal. BCE
2. El Vilot de
1412 cal. BCE
Montagut
888 cal. BCE
773 cal. BCE
1000 cal. BCE
3. Masada R.
896 cal. BCE
4. Tozal R.
Barley
δ13Cair
(‰) N δ13C (‰) ∆13C (‰) TWI (mm)
-6.36
2 -22.4 ± 0.0 16.4 ± 0.0 82 ± 0
-6.42
2 -22.2 ± 0.6 16.1 ± 0.6 76 ± 18
-6.47
2 -20.9 ± 0.2 14.8 ± 0.2 23 ± 2
-6.54
2 -22.9 ± 0.3 16.7 ± 0.4 86 ± 20
-6.54
2 -22.6 ± 0.1 16.4 ± 0.1 69 ± 5
-6.53
2 -21.6 ± 0.5 15.4 ± 0.5 58 ± 10
-6.54
2 -21.8 ± 0.1 15.6 ± 0.1 40 ± 3
5. Els Vilars
-6.54
-6.54
-6.59
-6.45
-6.45
-6.45
-6.36
-6.42
6. Margalef
7. R. Sarró
8. T. Tenalles
9. Lleida
9. Lleida
Total
793 cal. BCE
781 cal. BCE
500 BCE
200 BCE
200 BCE
200 BCE
300 CE
1025 CE
2
2
2
2
-
-22.3 ± 0.9
-21.8 ± 0.0
-21.1 ± 0.0
-23.4 ± 0.0
-
16.2 ± 1.0
15.6 ± 0.0
15.0 ± 0.0
17.4 ± 0.0
-
71 ± 40
40 ± 1
27 ± 0
120 ± 1
-
2
2
1
27
-23.0 ± 0.2
-22.2 ± 0.1
-22.6
-22.2 ± 0.2
17.0 ± 0.2
16.2 ± 0.1
16.5
16.1 ± 0.2
100 ± 15
76 ± 3
86
68 ± 6
N
2
2
2
2
2
1
-
δ13C (‰)
-22.4 ± 0.0
-20.8 ± 0.0
-21.7 ± 0.0
-22.1 ± 0.0
-22.2 ± 0.1
-22.1
-
Wheat
∆13C (‰)
16.4 ± 0.0
14.7 ± 0.0
15.6 ± 0.0
15.9 ± 0.0
16.0 ± 0.1
16.0
-
TWI (mm)
88 ± 1
47 ± 0
66 ± 0
73 ± 0
77 ± 2
75
-
2
1
2
2
2
2
2
1
25
-21.2 ± 0.1
-20.9
-20.4 ± 0.0
-22.0 ± 0.0
-21.6 ± 0.6
-21.9 ± 0.0
-23.0 ± 0.2
-23.0
-21.8 ± 0.2
15.0 ± 0.1
14.6
14.2 ± 0.0
15.9 ± 0.0
15.5 ± 0.6
15.8 ± 0.0
17.0 ± 0.2
17.0
15.7 ± 0.2
52 ± 1
46
40 ± 0
73 ± 0
65 ± 14
71 ± 0
110 ± 9
108
70 ± 4
134
 Chapter 6. Grain weight in archaeological cereal seeds: role of climate and genetic improvement
6.3.3 To what extent water stress conditioned
grain weight in ancient crops?
GW is a character with a strong genetic basis,
and thus its evolution with time might respond to
either genetic or environmental changes. Although,
in some cases, DNA can be recovered from
archaeological charred grains (see e.g. Brown
1999; Schlumbaum et al. 1998), systematic studies
of the genetic pool of such material are still not
feasible. Alternatively, we can quantify the
potential role of environmental conditions (at least
those related with water availability) by comparing
∆13C and GW evolution. Firstly, we took a subset
of 26 archaeological samples (14 for wheat 12 for
barley) in which pairwise comparisons between
both variables were possible. We found a weak,
but significant relationship between ∆13C and GW
for wheat and barley. The heterogeneity of slopes
18
16
13
∆ C (‰)
17
15
14
35
Wheat
Barley
a)
GW (mg)
30
25
20
15
10
Residual (mg)
6.3.2 Evolution of carbon isotopes and grain
weight during the last 4000 years
In general terms, wheat and barley showed
similar trends throughout the studied period for
∆13C (Fig. 6.2a). ∆13C was similar to current values
during the second millennium BCE, showing a
strong depletion from ca. 800 BCE to 500 BCE.
After this, a significant recovery is apparent from
ca. 300 BCE, probably spanning to the XIth century
BCE. From the XIth century to present times, wheat
showed certain decrease in ∆13C, whereas barley
maintained similar values from ca. 300 BCE to
present.
In a similar way, wheat and barley followed
nearly the same pattern for the evolution of GW
during the last four millennia (Fig. 6.2b). However,
such pattern is contrastingly different from that
found for ∆13C. Present GW (30-35 mg) was far
greater than the estimations obtained for
archaeological material, which barely exceeded the
20 mg threshold from ca 2000 BCE to 500 CE).
We found a relatively constant increment in grain
weight during the first millennium BCE, followed
by a steeper increase from ca. 500 CE to present
times.
5
20
15
10
5
0
-5
-10
-15
b)
c)
200 0 1500 10 00 500
cal.BC E
0
500 10 00 1500 2 000
BC E C E
Fig. 6.2 Evolution of a) carbon isotope discrimination (∆13C)
and b) estimated grain weight (GW) in wheat (filled circles)
and barley (empty circles) during the last four millennia. As a
reference, present values of ∆13C {Ferrio, Araus, et al. 2005
6491 /id} and grain weight {Ferrio, Alonso, et al. 2004 5677
/id} in the area are plotted. c) residuals resulting from the
linear regression between GW and ∆13C (Eq. 6.8). Trend lines
depict locally weighted least squares regression curves
(LOESS, span=0.4) fitted to the data. Vertical error bars
indicate the standard error of the mean.
ANOVA revealed that the slope of the resulting
regression lines did not differ significantly for the
two species, but they had distinct intercepts. Thus,
we assumed equal slopes for the two species, and
fitted the following model, including barley and
wheat data:
Eq. 6.8
GW = −40.7 + 3.53 × ∆13C + 4.6 × ( sp = wheat )
r 2 = 0.40, P < 0.01
where sp=wheat is an indicator variable which
takes the value 1 if true and 0 if false, in order to
modify the intercept of the regression, according to
To be submitted to Journal of Cereal Science 135
the species. We subsequently applied this model to
all the studied sites, and the residuals between GW
(calculated from grain dimensions) and GW
predicted by ∆13C were plotted (Fig. 6.2c). The
departure from the zero line of the residuals would
be an indicator of GW changes that are not
attributable to changes in water availability. The
most clear trend in the evolution of residual GW is
a great increase from archaeological to modern
material. Such increment might have been started
during Middle Age, but still during this period GW
values were significantly lower than in presenttimes. Whereas the residual for archaeological
grains oscillated around zero (± 5 mg), GW in
modern material was much higher than the
expected values derived from Eq. 6.8 (+19.0 mg
and +13.1 mg, for wheat and barley, respectively).
Indeed, the residual plot have enhanced the
observed increment in GW for modern crops,
which contrasted with the relatively stable ∆13C
values. At this point, we should consider the
possibility that grain weight in archaeological sites
was limited by environmental constraints others
than water availability. For example, both pre- or
post-anthesis temperature stress may decrease
strongly final grain weight (Calderini et al. 1999a;
Calderini et al. 1999b). However, under semi-arid
climates, higher temperatures should be also
translated into lower ∆13C in plants, due to an
increase in vapour pressure deficit (Condon et al.
1992; Ferrio and Voltas 2005; Zhang and Nobel
1996). This was not our case, as we found an
average ∆13C in archaeological sites (see Table
6.2) similar to that found in present crops (16.4‰
±0.5 for barley and 15.4‰ ±0.9 for wheat).
Moreover, several palaeoenvironmental evidences
indicate a global rise in temperature since mid
XIXth century (Barriendos and Martín-Vide 1998;
Creus et al. 1996; Folland et al. 2001; Riera et al.
2004), further discarding the possibility of higher
temperature stress in the past. Alternatively,
current agronomic practices, like the extensive
application of nitrogen fertilisers, might be also
responsive of changes in grain size. However,
although poor nitrogen status may be as important
as water scarcity in limiting crop performance in
Mediterranean climates (Passioura 2002), the
effect of nitrogen availability on grain weight is
less clear. Nitrogen starvation tends to decrease the
number of grains per plant, but has little effect on
individual grain weight (Oscarsom et al. 1999) and
in some cases can lead to greater grain weights,
due to compensation mechanisms (Corbeels et al.
1999; Ferrio et al. 2004b).
The apparent lack of environmental constraints
(at least in terms of water availability) underlying
the lower grain size of archaeological grains,
compared with modern material, suggest that it
was determined by genetic factors. Such genetic
gain in grain weight cannot be attributed, however,
to modern plant breeding programs, as traditional
landraces and modern cultivars show similar
values for this trait (Abeledo et al. 2002; Cascón
1934). Indeed, grain size might have been among
the first selection criteria used by farmers. Greater
grains show higher flour yield and lower bran
content, thus affecting their nutritive value (Henry
and Kettlewell 1996; Wiersma et al. 2001), and
also have better malting quality in barley (MolinaCano et al. 1997) and baking properties in wheat
(Evers et al. 1995). Moreover, grain weight is also
positively associated with germination power
(Mian and Nafzinger 1994) and early vigour
(López-Castañeda et al. 1996; Richards and
Lukacs 2002), ensuring a better development,
specially for the harsher environmental conditions
(Grieve and François 1992; Mian and Nafzinger
1994). Thus, it is quite probable that selecting the
biggest seeds for sowing was a well-known
practice among ancient farmers (Cascón 1934;
Zeven 2000).
6.3.4 Climatic and genetic effects on the water
status of cereal crops
To further assess the environmental
background in which archaeological crops were
developed, we compared the evolution of TWI in
cereals with estimated annual precipitation (Fig.
6.3), derived from ∆13C analyses of charcoal
fragments of Aleppo pine (Pinus halepensis Mill.),
recovered in the same sites (Ferrio, Alonso, López,
Araus and Voltas, submitted manuscript). The
climatic reconstruction derived from forest trees
allows us to determine the role of general climatic
136
 Chapter 6. Grain weight in archaeological cereal seeds: role of climate and genetic improvement
800
D?
H
D
H
D?
H? D
Pan (mm)
700
600
500
400
Cereal TWI (mm)
300
200
120
a)
100
80
60
40
20
0
b)
2000 1500 1000 500
cal.BC E
0
500 1000 1500 2000
BC E C E
Fig. 6.3 Comparison between the evolution of estimated mean
annual precipitation (Pan, Ferrio et al., submitted) and total
water inputs during grain filling (TWI) in cereals (wheat and
barley combined) in the studied area. Pan was estimated from
carbon isotope discrimination (∆13C) of Pinus halepensis
charcoal in the same sites used in the present work. Trend
lines depict locally weighted least squares regression curves
(LOESS, span=0.3 for Pan, span=0.4 for TWI) fitted to the
data. Relatively dry (D) and humid (H) phases (according to
Ferrio et al.) are indicated, as well as periods of strong
climatic variability (shadow areas).
trends on the observed changes in cereal TWI. Due
to the small amount of sample available, the period
from ca. 2000-1000 BCE was poorly characterized
for the cereals, and thus the results are not fully
comparable. Nevertheless, in both cases the driest
period, among archaeological samples, was placed
between ca. 800 BCE and 500 BCE, and it was
followed by a recovery from ca. 300 BCE to 300
CE. The subsequent period of strong climatic
variability around 1000 CE could not be contrasted
in the cereals, as only two samples of cereal were
analysed for this period. Intriguingly, whereas
estimates of annual precipitation in the past (from
wood ∆13C) were consistently higher than in
present-times, this was not the case for cereal TWI.
The most probable explanation for such divergence
could rely on the recent genetic change of cereals
resulting from plant breeding programs. Modern
cultivars have been usually selected to attain better
performance under optimal conditions (i.e. to
maximise yield potential), being less conservative
in terms of water expense (Araus et al. 2002). This
is translated into lower stomatal limitation of
photosynthesis, and thus higher ∆13C than
traditional landraces when growing under the same
conditions (Fischer et al. 1998; Zhao et al. 2001),
with differences of up to 1‰. Moreover, the
varieties released since 1960 are characterised by
an overall reduction in plant height and growing
cycle, and hence the grains are developed under
cooler and more humid conditions, contributing to
the increase ∆13C (Abeledo et al. 2002; Araus et al.
2002). Accordingly, it would be helpful to compile
more reference data from traditional landraces, in
order to make proper comparisons between
archaeological and present-times.
6.4. Conclusions
Our results confirm that, in the area of study,
grain weight was significantly smaller in the past
than in present-times. We found an overall trend of
increasing grain weight since ca. 1000 BCE,
becoming steeper from ca. 1000 CE to present
times. According to ∆13C data, the most recent
increase in grain weight was not associated to
increased water availability. Consequently, we
concluded that the greater grain weight in presentday cereals responded to a genetic improvement of
the crops, which might have started sometime from
Roman Period. The general agreement between
estimations of annual precipitation, derived from
fossil charcoal, and estimated water inputs in
cereals, indicates that the water status of such
crops was mostly determined by climatic
constraints. Nevertheless, modern material used for
reference caused an overestimation of present
water status, probably due to the greater stomatal
conductance associated to modern varieties.
Acknowledgements
This work was partly supported by the CICYT grant
BTE2001-3421-C02-01 and the INCO-MED project
MENMED (ICA3-CT-2002-10022). J.P. Ferrio has a
PhD Fellowship from Generalitat de Catalunya.
To be submitted to Journal of Cereal Science 137
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 Chapter 6. Grain weight in archaeological cereal seeds: role of climate and genetic improvement
General Discussion
“The most merciful thing in the world, I think, is the inability of the
human mind to correlate all its contents.”
H.P. Lovecraft. The Call of Cthulu
141
General Discussion
I. Different roads to Rome: evolutionary keys for the divergences
between Aleppo pine and Holm oak in response to drought
The temporal uncoupling of temperature and precipitation, with the driest period coinciding with the
highest temperatures, as well as the existence of nutrient-deficient soils have been major evolutionary
forces in shaping Mediterranean plant communities (Herrera 1992; Mooney and Dunn 1970). Examples
of common adaptations include extensive root systems, evergreen foliage to compensate for nutrient
scarcity and to permit year-round production, water-saving mechanisms such as leaf sclerophylly or a
very sensitive stomatal regulation (Rundel 1988; Zavala et al. 2000). The results presented in Chapter 1
confirm that, although both Aleppo pine (P. halepensis Mill.) and Holm oak (Quercus ilex L.) are well
adapted to Mediterranean climates, the strategies adopted by each species differ considerably. P.
halepensis is a typical drought-avoiding species, which concentrates most of their response to drought on
water saving (mostly through stomatal closure), but acts as an opportunistic species when conditions are
favourable. On the other hand, Q. ilex is a slow growing, deep-rooted species that, although showing a
considerable stomatal regulation (at least when compared to other species of the same genus), shows a
very effective water uptake, together with drought-tolerance mechanisms, such as osmotic adjustment.
This explains the strong response to water availability of carbon isotope discrimination (∆13C) and radial
growth in P. halepensis, contrasting with the weaker sensitivity of Q. ilex. The apparent lack of sensitivity
in radial growth of Q. ilex, however, contrasts with the results of studies of the temporal variability of
tree-rings (Corcuera et al. 2004). Over the great spatial gradient here studied, several compensation
mechanisms might have minimised long-term variability in radial growth: changes in phenology, different
patterns of assimilate distribution between leaves and xylem (Villar-Salvador et al. 1997). Such
compensation mechanisms might result from either acclimation processes (Villar-Salvador et al. 1998;
Villar-Salvador et al. 1999) or genetic differences between provenance regions (Gratani et al. 2003;
Tognetti et al. 1997).
∆13C values in Q. ilex, moreover to having lower sensitivity to water availability, were consistently
higher than those found in P. halepensis (see Fig. 1.3). Previous studies on co-occurring conifers and oaks
have also shown that the former tend to show lower ∆13C than the latter (Picon et al. 1996; Valentini et al.
1992). Differences in ∆13C between Q. ilex and P. halepensis, for the common range in the ratio between
precipitation and evapotranspirative demand (P/E = 0.45-1.08), ranged from 0.7‰ to 1.5‰, being higher
in drier environments. Thus, for the same levels of aridity, P. halepensis displays stronger stomatal
limitation of photosynthesis, and consequently higher water use efficiency (WUEi) and lower ∆13C, and
this difference increases in response to water stress. At this point, we should bear in mind that the
proportion of lignin and cellulose, as well as the metabolic routes for the synthesis of lignin, are different
in oaks and conifers, and this might cause differences in ∆13C between species, not related with WUEi
(Barbour et al. 2001; Borella et al. 1998), see also Chapter 2). Nevertheless, when comparing ∆13C in
holocellulose for the two species (see Chapter 2, and Vega 2003 for data on Q. ilex) we found the same
pattern observed for wood, and the absolute differences were even higher (from 0.8‰ to 1.8‰, over the
143
General Discussion
same range of P/E). All these results confirm that Q. ilex, despite being a species with a strong capacity of
stomatal regulation (Damesin et al. 1998; Infante et al. 1999), should be considered as a partially drought
tolerant species, which also relies on other mechanisms to cope with drought. Moreover, to be able to
extract water from far deeper soil layers, the threshold of predawn water potential for stomatal closure for
Q. ilex is much lower than for P. halepensis (Borghetti et al. 1998; Damesin et al. 1998). Consequently,
Q. ilex can show, over the growth season, lower stomatal limitation to photosynthesis than a droughtavoiding conifer such as P. halepensis. Our current work provides additional evidences on the
physiological differences between the two species, using combined analyses of carbon and oxygen stable
isotopes. According to the conceptual model proposed by Scheidegger et al. (2000), we hypothesized in
Chapter 2 that, in a species with a strong variability in stomatal conductance (e.g. P. halepensis), the
oxygen isotope composition in wood holocellulose (δ18OC) would be mostly associated with the water
status of trees, rather than with the temperature signal derived from the isotopic composition of rainfall
(δ18OR). Conversely, a species showing alternative strategies to cope with drought, as is the case for Q.
ilex, would display δ18OC more closely related to δ18OR. We have found that, unlike P. halepensis, δ18OC
in Q. ilex (over 15 of the sites described in Chapter 1) can be correlated with temperature (r= 0.58,
P<0.05) but not with vapour pressure deficit (Ferrio and Voltas, unpublished results). Moreover, no
relationship at all was found between δ13C and δ18O values in holocellulose for Q. ilex (r=-0.01),
contrasting with the weak, positive trend described for P. halepensis (r=0.36, P<0.1). Although δ18O
results for Q. ilex are still preliminary, they suggest that δ18OC variability across sites for this species
cannot be attributed to evaporative enrichment of leaf water, as happened in P. halepensis, being mostly
determined by source water. Again, this is a result of the lower stomatal control of Q. ilex, in relation to
P. halepensis.
Some of the abovementioned divergences might derive from the different evolutionary history of P.
halepensis and Q. ilex. According to Herrera (1992), life-history traits of Mediterranean species result
from a combination of responses to evolutionary processes acting on pre-Mediterranean lineages. Thus,
the opportunities to adopt a given adaptive strategy to Mediterranean-type climates are restricted by the
particular morphological and physiological traits of each evolutionary line. In extreme cases, certain
groups, too adapted to previous environmental conditions, disappeared from the Mediterranean area.
Other groups succeeded to develop new drought-adapted forms, but through mechanisms constrained by
their primitive traits. In the case of P. halepensis, it shares with other conifers the primitive xeromorphic
characters of the group (e.g. low surface/volume ratio of leaves), which appeared during the increase in
aridity occurring ca. 300 My BP, at Permian (Thomas and Spicer 1987). Moreover, the thermophil nature
and indefinite growth of P. halepensis might also be a primitive character from the sect. Pinea (including
P. pinea, P. tropicalis and P. brutia), which diverged from the sect. Sylvestres in Lower Cretacean, about
120 My BP (Klaus 1987). The former was located in the euro-caribbean region, growing under warm,
sub-humid to semi-arid climates, whereas the latter was restricted to higher, colder latitudes. The first
fossil appearance of P. halepensis occurs during the Eocene (50 My BP), a period characterised by an
increase in aridity around Southern Europe (Klaus 1987; Palamarev 1987). Thus, the evolutionary history
144

General Discussion
of P. halepensis is marked by successive environmental pressures towards an adaptation to dry, warm
climates. On the other hand, the potential ancestors of Q. ilex appeared at the end of the Upper Cretacean
(70 My BP) in the form of evergreen, laurophyllous species within the paleotropical geoflora, and
associated with warm, humid environments (Mai 1987; Manos and Stanford 2001). The first appearance
of distinctive traits of Q. ilex coincides with the increase in continentality that occurred during the
Oligocene (35 My BP), which might explain the presence of winter dormancy in this species (Mai 1987;
Palamarev 1987). Indeed, the first fossil remains of Q. ilex appear associated with a new type of
vegetation, the “mixed mesophytic forest”, together with deciduous species from arctoboreal origin (Mai
1987). During this period, Q. ilex was mostly found in continental environments, forming closed forest,
whereas P. halepensis occupied mostly open spaces in coastal areas (Barrón 2004), thus resembling
current habitat preferences (see Fig. VIII). Nowadays, P. halepensis is clearly better adapted than Q. ilex
a)
b)
Fig. VIII Relationship between climate and potential distribution of Quercus ilex L. and Pinus halepensis Mill. in Eastern Spain.
a) Main bioclimatic regions, according to the UNESCO humidity index (total precipitation/Pemman evapotranspirative demand),
and isotherm of 5ºC from January average temperature. Adapted from Spanish Ministry of the Environment (2000) and IGN
(1992). b) Climacic distribution of the two species. Adapted from IGN (1995).
145
General Discussion
to the typical (semi-arid) Mediterranean climate of the Iberian Levant and Southern Mallorca, but is
rapidly replaced by the latter in wetter environments favouring the formation of dense, dark forests (e.g.
Northern Catalonia, Western Mallorca), as well as in the continental Mediterranean climates of the
Central Plateau. In such context, some potential adaptations of Q. ilex to drought might originally come
from the need to reduce the effect of fire events and hervibores, rather than from a drier climate (e.g. hard,
sclerophyllous leaves, deep-roots to allow sprouting). Summarising, the evolutionary history of the two
species indicates that the great response to drought of P. halepensis is derived from successive selection
processes, associated with dry episodes, whereas for Q. ilex the adaptation to Mediterranean-type climates
is relatively recent, and probably linked to other environmental and/or biotic constraints.
II. Sensitivity to seasonal climate of stable isotopes in tree-rings:
implications for (palaeo)environmental studies
Throughout the year, the periods in which precipitation events have a significant effect on the isotopic
signal of wood differ substantially between Q. ilex and P. halepensis. In the case of P. halepensis, the
good sensitivity of ∆13C to changes in precipitation nearly during the whole year is compatible with the
indeterminate growth of this species (Liphschitz and Lev-Yadun 1986; Nicault et al. 2001). Indeed,
cambium activity in P. halepensis can span over the whole year under favourable conditions, although
can be slowed during the driest weeks of summer, as well as in cold winters. Indeed, we found the lowest
correlations between monthly precipitation and ∆13C in January and July, coinciding with the coldest and
the driest month of the year, respectively (see Fig IXa). Thus, for P. halepensis, the periods of greater
sensitivity to climate variables coincide with the expected periods of active growth. This seems to be the
case also for other conifers. Indeed, the seasonal pattern of isotopic composition in wood of Pinus species
has shown to be mostly determined by the environmental conditions in which each wood layer was
formed (Barbour et al. 2002; Jäggi et al. 2003; Leavitt and Long 1991; Schulze et al. 2004). For Q. ilex,
however, the seasonal variation in the sensitivity of bulk wood material to precipitation contrasts with the
phenology of this species. Unlike P. halepensis, Q. ilex has a true dormancy period during winter, as well
as a period of reduced growth in summer (Liphschitz and Lev-Yadun 1986). Interestingly, the time in
which precipitation has the greater effect on the ∆13C of wood correspond in part with the period of winter
dormancy. Winter rains, although occurring during cambium dormancy, can feed deep-soil water
reservoirs, which in turn increases effective water availability during the growing period for a deeprooting species, such as Q. ilex. On the other hand, we cannot rule out the possibility that early wood
formation during spring could rely on the remobilisation of photosynthates synthesised during autumn
and early winter. Remobilisation of stored carbohydrates is a necessary feature in deciduous trees, as
many of them start wood growth before leaf emergence. Recent studies on radial sub-sections of treerings have shown that this process may alter significantly the isotopic signal of early wood, precluding its
use as an environmental indicator (Helle and Schleser 2004). However, there is a lack of such kind of
studies on evergreen, sclerophyllous species such as Q. ilex. Currently, we are preparing a study to
compare the isotopic seasonal pattern in different co-occuring species from the genus Quercus, covering
the existing range of leaf habits, from the evergreen Q. ilex to the deciduous Q. petraea, going through
the marcescents Q. faginea and Q. humilis. The objective of this work will be to find out whether such
146

General Discussion
“isotope anomaly” of early wood is limited to deciduous trees, or it is also present in evergreen species
from the genus.
Despite the aforesaid limitations, the differences in the response to seasonal rainfall between species
should not be considered only as a handicap for the use of stable isotopes in tree-rings as
(palaeo)environmental indicators. Quite the opposite, it is an interesting feature that, provided a good
physiological knowledge of the studied material, can provide additional environmental information by
combining data from different species. In our case, P. halepensis appears, at first glance, as the most
suitable species to attain climatic inferences from wood ∆13C, as it is sensitive to precipitation events
occurring throughout the year. However, if we compare the response to seasonal rainfall in stands of P.
halepensis and Q. ilex within the same geographic area, the divergences between the two species can be
used as an indicator of changes in seasonality. For example, as P. halepensis is sensitive to winter colds,
and has a poor ability to exploit winter-fed water reserves in the soil, an increase in winter precipitations,
accompanied with a descent in temperature, would not cause an increase in ∆13C. Indeed, it might cause a
decrease in bulk ∆13C, as a greater portion of wood is synthesised during spring/summer. In contrast, Q.
ilex would be clearly favoured by this situation: the augment in deep-soil water would increase effective
water availability for Q. ilex, leading to an increase in the ∆13C of wood. Thus, under such situation, we
would find relatively high ∆13C in Q. ilex, but not in P. halepensis (see Fig. IXb). Similarly, an increase in
summer storms would cause a positive response in the opportunistic P. halepensis, while having little
effect on Q. ilex. Such kind of conceptual approach might be useful to clarify the real nature of certain
climatic anomalies found in the palaeoenvironmental record: the next step would be the transformation of
this conceptual model into a quantitative one (calibrated with data from mixed forests), in order to
maximise the information obtained from the two species.
Fig. IX a) Comparison of the determination coefficients (r2) between carbon isotope discrimination (∆13C) in wood and monthly
precipitation for Quercus ilex and Pinus halepensis (data from Chapter 1). b) Proposed conceptual model for the interpretation of
∆13C values in co-occuring Q. ilex and P. halepensis, according to their differential response to seasonal precipitation. ∆13CPinus,
∆13CQuercus, ∆13C in wood of P. halepensis and Q. ilex, respectively; Pspring, Psummer, Pautumn, Pwinter; total seasonal precipitation; ↑,
increasing variable; ≈, steady variable; ↓, decreasing variable; α = 0.05, threshold line for significance at the 5%.
147
General Discussion
Reinterpreting the “Cold Iron Age Epoch” in the Ebro Basin on the light of ∆13C data
We can take advantage of this conceptual model to further discuss the environmental conditions in the
Segre and Cinca Valleys during the so called Cold Iron Age Epoch (ca. 900-300 BCE). (Gribbin and
Lamb 1978) defined this period, estimating a decrease in temperature of about 2ºC, evidenced by an
expansion of the glaciers, among other evidences of an abrupt climate cooling in Europe (VanGeel et al.
1996). According to the ∆13C analysis of P. halepensis charcoal (Chapter 3), this period appears as a
relatively arid phase in the Segre and Cinca Valleys. Such increase in aridity is confirmed by pollen data
from El Vilot de Montagut (Alonso et al. 2002), and other sites from Catalonia (Riera 1994), as well as
hydrological studies from NW Mediterranean (Magny and Richard 1992). However, other
palaeoenvironmental proxies provide (apparently) contradictory evidences for this period.
Geomorphological studies performed throughout the Ebro Basin (Gutiérrez-Elorza and Peña-Monné
1992) indicated a generalised increase in sediment accumulation on slope deposits around 1000-400 BCE,
which was interpreted as an increase in vegetation cover, and a colder/wetter climate. Moreover, pollen
analyses from a salt lake in Central Ebro Depresion (Salada Pequeña) suggested an increase in water
levels around 2600-2000 BP (650-50 BCE) (Davis 1994). Are these evidences compatible with a water
shortage in P. halepensis stands?. The answer to this question might come from the role of seasonality.
Anthracological evidences from the Cinca and Segre Valleys (Alonso 1999; Ros 1997), show the
existence of mixed forests of holm oaks, pines and deciduous oaks, whereas for the Central Ebro
Depression the same studies evidenced a decrease in vegetation cover, and increased continentality (Ros
1997). Thus, and according to the adaptive traits discussed in section I, the spread of evergreen and
deciduous oaks in the area could be interpreted as a result of an increase in continentality, rather than a
consequence of a wetter climate. Although δ13C values in oak from Southern France (Vernet et al. 1996),
see Fig. 4.3) also indicated an arid episode ca. 3000-2700 BP (1050-750 BCE), it was followed by a
recovery in δ13C values from ca. 2600-1600 BP (650 BCE-350 CE), more than three centuries before the
recovery of P. halepensis (ca. 300 BCE). The same pattern has been recently confirmed by δ13C analysis
of oak charcoal (both evergreen and deciduous) recovered from the same archaeological sites included in
Chapter 3 (Alonso et al., unpublished results). According to Fig. IXb, such divergence between pines and
oaks could be interpreted as an increase in winter precipitation, along with a decrease in spring-summer
rainfall. Indeed, ∆13C analyses of cereal grains (see Chapter 6), indicated that water inputs during grain
filling (late-spring) were significantly reduced during this period in the area, and also in other sites of NE
Spain (Araus et al. 1997; Araus and Buxó 1993; Ferrio et al. 2005a). On the other hand, as deciduous
oaks would be less adapted than evergreen oaks to summer drought, a decrease in summer precipitation
would explain the relative increase of the latter reported by Jalut et al. (2000). The observed increase in
lake levels (as well as glaciers) could be derived exclusively from increased winter recharges. Similarly, a
reduction in soil erosion might arise from the absence of convective storms (Gutiérrez-Elorza and PeñaMonné 1992), together with an increase in deep-rooted, cold-resistant species.
148
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General Discussion
III. Time course of CO2 during the Holocene and the use of carbon isotopes
in plant material as palaeoenvironmental tools
According to ice-core data (http://www.ncdc.noaa.gov/paleo/icecore.html) recent changes (i.e. during
the last 150 years) in atmospheric CO2 composition exceed those attained in the last half a million years.
Thus, we should consider this when using present ∆13C-climate relationships to reconstruct water
availability from the archaeological record, as recent increases in CO2 partial pressure might have a direct
effect on ∆13C values. Increasing ambient CO2 (Ca in Eq. IV) tends to decrease stomatal conductance, but
also stimulates CO2 assimilation rate through increased substrate supply for carboxylation (Farquhar et al.
1982). Bearing in mind that the relationship between water availability and ∆13C is mostly driven by the
stomatal limitation of the intercelular CO2 concentration (Ci), significant changes in Ca might alter such
relationship. Studies on tree-rings (Bert et al. 1997; Duquesnay et al. 1998; Saurer et al. 2004) and
herbarium material (Beerling and Woodward 1993; Friedli et al. 1986; Pedicino et al. 2002; Peñuelas and
Azcón-Bieto 1992) have shown a decrease in ∆13C from pre-industrial to present times, although this
trend varied considerably among species. Thus, for example, in the work of Peñuelas and Azcón-Bieto
(1992), the lack of a clear trend for ∆13C in P. uncinata (potentially more sensitive to Ca changes due to
its mountain habitat) contrasts with the negative drift of P. pinea, and the upward trend of Alnus
glutinosa. On the other hand, we should bear in mind that, during this period, the raise in Ca comes
together with an increase in global temperature, and increased aridity in many areas (Folland et al. 2001).
Thus, in this kind of retrospective studies, it is difficult to discriminate between Ca and climate effects on
∆13C. Interestingly, experimental studies under controlled environments have shown that ∆13C of a variety
of C3 plants suffer little change over a wide range of CO2 partial pressures (Beerling and Woodward
1995; Picon et al. 1996; Picon et al. 1997; Polley et al. 1993). In most cases, the increase in assimilation
rates was compensated by a decrease in stomatal conductance, thus minimising variations in the ratio of
intercellular to atmospheric partial pressure of CO2 (Ci/Ca) and ∆13C. Moreover, the relationship between
stomatal conductance and plant water status is comparable under different CO2 concentrations (Bunce
2004; Picon et al. 1996; Picon et al. 1997). Overall, these results indicate that ∆13C can be useful to
estimate past water availability, regardless of atmospheric CO2 changes. In addition, the variability of Ca
associated to the (unknown) canopy density in which archaeological remains were grown might exceed
recent changes in CO2 partial pressure in the atmosphere. This drive our attention to the need of a good
amount of replicate samples to avoid an artificial biasing of ∆13C values (due to variable amounts of
refixation of isotopically depleted CO2 from respiratory processes), but also implies that observed
differences between archaeological and present material can be hardly attributed to a “CO2 effect”.
Nevertheless, the variety of long-term physiological and ecological responses observed in plant systems
against CO2 increase still remains to be fully understood (Körner 2003), and thus there is still some
degree of uncertainty in past climate inferences based on the analysis of plant material.
149
General Discussion
IV. Reconstruction of plant growing conditions in ancient crops:
main limitations and future prospects
Overall, the aim of Chapters 4 to 6 was to find alternative approaches to maximise the information
about ancient crops that can be derived from the study of archaeobotanical remains. We have shown that
combining stable isotope analyses from different species can be useful to find evidences of water
management practices. Similarly, the existence of potential genetic changes in cereals can be assessed by
relating ∆13C and grain weight estimates. However, there are still some questions that should be
considered, in order to obtain the most reliable picture of ancient crops. It should be noted, for example,
that current models to estimate water availability in ancient crops have been developed from modern
material, grown under conventional agronomic trials. Thus, the application of such models on
archaeological material assumes implicitly that no significant changes in ∆13C can be derived from recent
genetic and/or agronomic changes. Another important topic, specially for grain weight estimations, is the
origin and amount of the samples analysed, as discussed below.
To what extent are grain weight estimations a reliable source to study
changes in potential (i.e. genetic) grain size of ancient crops ?
Grain weight was probably the earliest genetic trait consciously improved by prehistoric farmers, as
already stated in several early studies on agriculture, which drove attention to the traditional selection of
the biggest grains for sowing (Eguaras 1988; Wilkinson 1969; Zeven 2000). Unexpectedly, our work
suggest that, in the Segre and Cinca valleys, little genetic improvement in grain weight occurred until the
last two millennia, but these results should be taken with caution. Firstly, we cannot generalise these
findings to other regions. In general, values of grain dimensions from other sites are similar in range to
those here reported (Buxó 1997; Colledge 2001; Lone et al. 1993; Willcox 2004). However, whereas
Willcox (2004) has evidenced an increase in grain size throughout the earliest phases of agriculture in the
Middle Euphrates, Buxó (1997) did not found any clear trend in grain dimensions since Early Neolithic to
Middle Ages, across a wide range of sites from East Iberian Peninsula. On the other hand, as already
mentioned in Chapter 5, another inherent risk of our approach is that, except for the sites of Margalef and
Tossal de les Tenalles (Second Iron Age, ca. 200 BCE), carbonised remains used in our study did not
came from storage elements, and thus might not be representative of the material harvested by farmers.
Thus, in most cases we cannot rule out the possibility that the grains were consciously discarded by them.
Nevertheless, the grains from these two sites, recovered from storage jars, are still significanty smaller
than current material, and similar in size to those found in other archaeological contexts (domestic fires,
cooking ovens, room floors, levels of rubble). Moreover, the ∆13C of seeds from these sites suggests that
the environment in which were grown was even wetter than today, further supporting the existence of a
genetic constraint to grain size. Consequently, all indicates that, in the studied area, a considerable change
in the genetic potential for grain weight has occurred in the area, although not before that during the last
two millennia. Unfortunately, the amount of sample available for this period is too small, and we cannot
state definitely the starting point for such change. We are now preparing the analysis of new material
from Roman and Middle Age sites (Alonso 2005), in order to obtain a more robust assessment of ∆13C
150

General Discussion
and grain weight evolution for the last two millennia. One key question that still remains unsolved is
whether the Romanisation process already included the implementation of “improved” varieties (i.e. with
greater grain size) in this area, or the onset of current landraces is more recent. A complementary source
of information for this period might come from documentary studies, e.g. taking advantage of the fact that
cereal grains have been traditionally used as standards for weight and length measurements
(http://www.metrum.org/measures/metrics.htm).
Genotype effects on water availability and ∆13C
Nowadays, ∆13C is considered as a polygenic trait showing a strong heretability in several crops. In
wheat, for example, broad sense heritabilities (proportion of total variance related to genotypes) ranges
from 60 to 90% (Araus et al. 1998; Ferrio et al. 2005b). Similar results have been found in barley (Voltas
et al. 1999), peanut (Hubick et al. 1988), common bean (Ehleringer et al. 1991), and many other C3 crops
(Hall et al. 1990; Hall et al. 1994). Thus, although environment is by far the main factor responsive for
∆13C variations in C3 plants, the role of genotype should not be neglected. Although most ∆13C studies
have been performed on modern varieties, the few including traditional landraces have shown that they
tend to show lower ∆13C values than improved cultivars, mainly due to changes in growing cycle duration
(Fischer et al. 1998; Zhao et al. 2001). Consequently, the genetic differences between archaeological
crops and modern reference material might have led to an underestimation of water availability in the
past. Changes in phenology, and their effect on yield components and ∆13C, are currently under study,
using traditional landraces grown together with modern cultivars. This would provide a more reliable
reference value for comparison with ancient crops, as well as to provide clues about the causes for such
divergence.
Traditional practices and water availability of crops
Traditional agronomic practices (related with water harvesting, sowing and soil conditions) may
provide more water to rainfed crops than modern techniques (Capillon and Séguy 2002; Raupp and König
1996). Thus, opposite to the potential effect of genetic changes, the use of ancient agronomic practices
would cause higher water availability (and thus higher ∆13C) in ancient crops. For example, ploughing
appear to be a very ancestral practice, well documented in Mesopotamia from the fourth millennium
BCE, and probably known in Europe from the end of Neolithic or, at the latest, early Bronze Age, being
extensively used during the Iberian period (Civil 1994). Early ploughs were entirely of wood, later
including metal shares, causing substantially less soil disturbance than modern techniques of soil
preparation. After ploughing, seeds were usually sown by hand to a given deep in the soil Thus, ancient
ploughing and sowing practices would probably favour the preservation of soil water (Capillon and Séguy
2002). This is partly confirmed by the results from multifactor field trials performed at the Universitat de
Lleida, and including different water regimes, nitrogen supplies, and sowing techniques (Cantero et al.,
unpublished results). According to this experiment, the use of direct sowing (less aggressive, closer to
ancient labour works than standard ploughing) results in significantly higher ∆13C, specially in the most
favourable environments. Following these results, we are currently compiling material from similar trials
performed in other areas, with the aim to refine current models of water availability.
151
General Discussion
Considering other environmental factors: nutrient availability and temperature
In Mediterranean environments, rainfed cereal agriculture is not only limited by water shortage, but
also by the scarcity of nutrients in the soil (Cantero et al. 1995; Corbeels et al. 1999; Oscarsom et al.
1999; Passioura 2002), and the occurrence of high temperatures during grain filling (Calderini et al.
1999a; Calderini et al. 1999b; Ouabbou and Paulsen 2000).
Some agricultural practices, such as the addition of organic fertiliser or crop rotation with legumes can
leave a characteristic isotope signature in crop plants. As nitrogen isotope composition (δ15N) increases
with trophic levels, the addition of animal-derived nitrogen (e.g. manure or plant compost) tend to
increase the δ15N of plants (Bergstrom et al. 2002; Choi et al. 2003; Riga et al. 1971). In contrast, crop
rotation with legumes, usually having lower δ15N due to the fixation of atmospheric nitrogen (Shearer and
Kohl 1988; Yoneyama et al. 1990), is expected to give lower δ15N values (Högberg 1997; Riga et al.
1971). δ15N is also affected by water availability and indeed it is often correlated with δ13C (Handley et
al. 1999; Heaton 1987). Thus, although the fractionation processes affecting δ15N in plant matter are
complex (Choi et al. 2003; Hobbie et al. 1999; Högberg 1997), combining δ13C and δ15N analyses in
archaeological grains offer the potential to provide information about the nutritional status of crops.
Currently, we determine routinely δ15N in archaeological seeds, along with δ13C analyses. In most cases,
archaeological grains have higher δ15N than modern material, suggesting a systematic use of manure,
even in Neolithic sites. However, these conclusions are only temptative, due to the scarcity of reference
data regarding δ15N of plants under traditional crop regimes (i.e. without inorganic fertilisers).
The relationship between temperature and the oxygen isotope composition (δ18O) of precipitation has
been well established and is the basis for many palaeoenvironmental studies (see Introduction). In plants,
however, we must consider the isotopic enrichment occurring during transpiration, which is specially
important under Mediterranean environments (see e.g. Chapter 2). In Chapter 2 we have seen that both
δ13C and δ18O in wood cellulose of P. halepensis are, to some extent, affected by vapour pressure deficit
(VPD) and water availability. However, whereas δ13C is more sensitive to changes in water availability
than to VPD, the opposite is the case for δ18O. Bearing in mind that VPD integrates both humidity and
temperature information, the combined use of δ13C and δ18O would be of great interest in climate
reconstructions. Unfortunately, the need for cellulose extraction prevents the application of δ18O in wood
charcoal. The relatively homogeneous nature of cereal grains offer the possibility to be an alternative
material for the application of δ18O isotopes for palaeoenvironmental reconstruction. Indeed, most of the
aforesaid considerations regarding δ13C and δ18O in cellulose might be valid for cereal grains (Barbour et
al. 2000; Ferrio et al. 2005b; Pande et al. 1995). However, the amount of data available about the effect
of environmental variables on the δ18O of cereal grains is still scarce. Some preliminary results for the
Mediterranean Region (Fig. X) indicate that combining δ13C and δ18O analyses in cereal grains might
provide information not only about water availability, but also about temperatures during the growing
cycle. Nevertheless, it remains to be tested the effect of carbonisation on δ18O, which can be considerable,
due to the important oxygen losses occurring during this process (Kollmann 1955). We are currently
performing isotope analyses on cereal grains provided by the GENVCE Spanish winter cereal trial
152

General Discussion
network, in order to study the environmental signal that can be retrieved from the combined use of δ13C,
δ15N and δ18O, as well as the effect of carbonisation on δ18O. Moreover, we have determined average
grain weight for every sample analysed, and information on grain yield and other agronomic variables is
publicly available for these assays. With these data we aim to develop new multi-proxy models to infer
crop growing conditions and yield in ancient agriculture.
40
40
Grain δ18O
Grain ∆13C
35
35
30
30
r ²=0.66*
25
25
20
‰
‰
r ²=0.85**
20
15
r ²=0.59*
15
r ²=0.05
10
10
0
50
100
150
200
250
10
Water inputs (mm)
12
14
16
Average temperature (ºC)
Fig. X Relationship between carbon isotope discrimination (∆ C) and oxygen isotope composition (δ18O) in wheat grains
and either total water inputs during grain filling and average temperature throughout the growing season, across nine field trials
located in Catalonia (3), Andalusia (3) and NW Syria (3). Data compiled from Araus et al. (2003), Ferrio et al. (2005b) and
Villegas et al. (unpublished results). *P<0.05 **P<0.01
13
153
General Discussion
V. Concluding remarks
The application of stable isotope techniques to environmental archaeology is still in its beginnings.
Since the pioneer works of Araus and Buxó (1993) and February and Van der Merwe (1992), many
advances have been made in the understanding of crop and forest ecophysiology, while the interest of
archaeologists in botanical remains has increased notably, to the point that archaeobotanical studies are
routinely performed in most archaeological excavations. The ultimate aim of this thesis was to expand the
amount of information that can be retrieved from archaeological plant remains. The development of a
methodology to obtain reliable estimations of past precipitation regimes from the analysis of wood
charcoal might contribute to the knowledge of past climate changes, moreover to being an additional
source of environmental data for archaeologists. This methodology would be hopefully expanded to other
species, as well as to other areas in the world, where information on past water availability is difficult to
attain through other methods. In this context, the EC project MENMED, in which we are currently
involved, is giving promising results for the Near East. On the other hand, the required calibration of the
climate effects on stable isotopes in extant wood material has also some interest per se, as Mediterranean
trees are still the “poor brother” of stable isotope studies, and they might help to understand the particular
behaviour of these species. Finally, we have proposed some new approaches regarding the reconstruction
of the environmental conditions in which ancient crops were grown, such as the objective comparison of
data from crop and forest species, as well as from different crops, or the quantification of grain weight.
They are promising tools, but are still in a preliminary phase, and surely they will require further
refinement. To conclude, the works here presented constitute just another piece in the complex puzzle of
the origin and spread of agriculture. In this context, stable isotope techniques can help to obtain a clearer
picture of the environmental constraints faced by early farmers. It is unlikely, however, that any technical
advance (apart from H.G. Wells’ time-machine) would provide a complete answer for the most intriguing
question on the origins of agriculture, that is the reason that pushed hunter-gatherers to initiate such a
self-defeating life-style. In words of a Bushman (Lee and DeVore 1968):
“Why should I farm when there are so many mongongo nuts?”
154

General Discussion
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160

Conclusions
“El bambú florece una vez cada cien años,
el ciruelo florece una vez al año,
el pino permanece siempre verde”
161
Conclusions
I. Reconstruction of climate
Ecophysiological studies on extant trees
I.1 Aleppo pine (Pinus halepensis) is more sensitive to water availability than holm oak (Quercus
ilex), showing a steeper raise in δ13C with increasing aridity. This confirms our hypothesis that a
partially drought-tolerant species, such as Q. ilex, would show lower increase in intrinsic water
use efficiency (WUEi) under drier conditions than a drought-avoiding species (e.g. P.
halepensis).
I.2
δ18O in holocellulose extracted from P. halepensis was mostly associated to variations in vapour
pressure deficit, being unrelated to the δ18O of precipitation. This confirms our hypothesis that,
in a species with strong stomatal regulation, the signal of source water (δ18OR) would be lost due
to the great variability in evaporative enrichment at the leaf level.
Climatic inferences from modern wood
I.3 Q. ilex and P. halepensis respond to a decrease in water availability by increasing their WUEi,
as inferred from the negative relationship between carbon isotope composition (δ13C) in wood
and both precipitation and the ratio between precipitation and evapotranspiration. Hence, in
these species, δ13C can be used as an indicator of water availability for (palaeo)environmental
studies.
I.4
For P. halepensis, δ13C in whole wood provides similar relationships with climatic variables as
those obtained with holocellulose. Indeed, the climatic signature of carbon isotopes is also
present in lignin, the other major component of wood. Thus, it is not required to purify
holocellulose to use δ13C of wood from P. halepensis as a (palaeo)environmental proxy.
I.5
For P. halepensis, holocellulose purification is however necessary to obtain consistent
relationships between oxygen isotope composition (δ18O) and climatic variables, at least when
working with the usual climatic ranges found in tree-ring studies. This is caused by the presence
of a strong variability in lignin δ18O, not associated to climatic variables.
I.6
For P. halepensis, δ18O in holocellulose, as δ13C, is related to precipitation and vapour pressure
deficit. However, the information from the two isotopes is complementary, as each one respond
in a different manner to these environmental variables, and the two isotopes are poorly
correlated.
I.7
The particular adaptive properties at the species level strongly determine the kind of information
provided by δ13C and δ18O. Thus, a careful selection of the material of study (e.g. taking cooccurring species with contrasting physiological performance) would probably increase
considerably the environmental information obtained from stable isotopes in tree-rings.
163
Conclusions
Stable isotopes in fossil charcoal and palaeoclimate
I.8 Although δ13C in wood varies considerably after carbonisation, the environmental signal of
wood δ13C is well preserved after carbonisation, and the shifts in δ13C caused by this process
can be successfully removed.
I.9
The study of δ13C in charcoal from P. halepensis, recovered in archaeological sites from the
Cinca and Segre Valleys (Ebro Depression), indicate that average precipitation (and the ratio
between precipitation and evapotranspiration) was about 30% higher than in present-times from
ca. 2000 BCE to XVIIIth century. Hence, present-day semi-arid conditions may be mostly due to
recent climatic shifts towards a harsher climate, probably enhanced by anthropogenic
disturbance.
I.10 There were two main phases of increased precipitation, (1500-900 BCE; 300 BCE-300CE),
alternating with phases of lower water availability, relatively closer to current conditions (20001500 BCE; 900-300 BCE). A period of strong climatic variability was identified from the tenth
to the eleventh century, followed by relatively humid conditions from the sixteenth to the
eighteenth century.
I.11 By combining δ13C analyses from species with different growing cycles and water management
strategies, such as P. halepensis and Q. ilex, it may be feasible to obtain additional climatic
information on the seasonality of precipitation events.
164

Conclusions
II. Reconstruction of crop conditions
II.1 Naked wheat (Triticum aestivum/durum) and hulled barley (Hordeum vulgare) were generally
grown under better water status in the past than in present-times. This is valid for cereals
cultivated in NE Iberian Peninsula (from ca. 5800 BCE to XIth century), SE Iberian Peninsula
(ca. 4500-300 BCE), as well as in the Fertile Crescent (ca. 7900-6500 BCE). In NE Iberian
Peninsula, the general agreement between estimations of annual precipitation, derived from
fossil charcoal, and estimated water inputs in cereals, suggests that the water status of cereal
crops was mostly determined by climatic factors, rather than by agronomic practices.
II.2 In some of the sites from NE and SE Iberian Peninsula, wheat was probably favoured by ancient
farmers, sowing it in better soils than barley, or under supplementary irrigation. Our results also
indicate that grain legumes (Vicia faba and Lens sp.), were usually grown under more humid
conditions than cereals, most likely in small irrigated plots.
II.3 There is considerable change in dimensions and morphology of wheat and barley grains during
carbonisation, but the products Length × Breadth and Length ×Thickness are relatively stable in
a wide range of carbonisation environments. From these products (measurable in archaeological
grains) it is possible to obtain reliable quantifications of grain weight in ancient cereal crops.
II.4 Within the Cinca and Segre Valleys, grain weight was significantly smaller in the past (from ca.
2000 BCE to XIth century) than in present-times. There was an overall trend of increasing grain
weight since ca. 1000 BCE, becoming steeper from Middle Age to present times. According to
δ13C data, the most recent increase in grain weight was not associated to increased water
availability. Thus, the greater grain weight in present-day cereals respond to a genetic
improvement of the crops, which might have started sometime from Roman period.
165
List of tables and figures
167
List of tables and figures
Table index
Table description
Page
Table I Standards, notation, abundance, observed range in plants and mean analytical error of the
stable isotopes most commonly used in plant physiology
18
Table 1.1 Main climatic parameters for the provenance regions of Q. ilex and P. halepensis
included in Chapter 1.
53
Table 1.2 Across-sites correlation coefficients between annual means of climatic variables and
either ∆13C or annual radial growth (mm) of Q. ilex and P. halepensis.
56
Table 2.1 Site description and oxygen isotope composition in precipitation (δ18OR) for 10 reference
sites used to assess the accuracy of vapour pressure and δ18OR models.
69
Table 2.2 Site description and mean values for δ13C and δ18O in whole wood, extracted wood,
holocellulose and lignin.
70
Table 2.3 Correlation coefficients among site means of δ13C and δ18O values for each of the wood
fractions studied.
71
Table 2.4 Correlation coefficients between site parameters and either δ C or δ O in each wood
fraction, as well as with dry-weight lignin to cellulose ratio.
73
Table 2.5 Stepwise linear regression models to predict annual and seasonal means of climate
variables from δ13C and/or δ18O in different wood fractions.
75
Table 3.1 Main geographical and environmental variables of sampling locations for reference
wood cores.
84
Table 3.2 Main geographical and environmental variables (present values), dating and cultural
period of the archaeological sites.
85
Table 3.3 Analysis of variance for the effect of carbonisation temperature and geographic origin in
δ13C and %C of wood.
86
Table 3.4 Raw δ13C of charcoal from archaeological sites, and present carbonised wood from
locations near the study area. Estimated δ13C and ∆13C of original wood.
87
13
18
Table 4.1 Ages, chronological date, ∆13C in grains and estimated water inputs during grain filling
for the archaeological samples analysed.
102
Table 4.2 Average values of ∆13C in wheat, barley and faba bean grains grown under rainfed and
irrigated conditions in the three geographic areas studied.
104
169
List of tables and figures
Table description
Page
Table 5.1 Mean and range for grain weight, dimensions and morphological indices before and after
experimental carbonisation.
117
Table 5.2 Effect of carbonisation conditions on weight and grain dimensions, and r2 of the linear
relationship between initial grain weight and grain dimensions after charring.
118
Table 5.3 Site description (location, dating, cultural period) and number of archaeological grains
used in the case study.
121
Table 6.1 Dating, cultural period, average grain dimensions and estimated grain weight for the
archaeological sites.
133
Table 6.2 Dating, estimated δ13C in atmospheric CO2, average δ13C and ∆13C in grains, and
estimated water inputs during grain filling of archaeological grains.
134
170

List of tables and figures
Figure index
Figure description
Page
Fig. I Range of δ13C within the main compartments of environment and biosphere.
19
Fig. II Seasonal and interanual evolution of δ13C of atmospheric CO2 in Northern Hemisphere,
Equatorial Belt and Southern Hemisphere.
20
Fig. III Simplified scheme of the relationship between ∆13C in C3 plants and stomatal conductance.
22
Fig. IV Main fractionation steps and typical values of δ18O in a temperate climate.
25
Fig. V Example to illustrate the construction of a tree-ring chronology.
30
Fig. VI Climate changes in central Greenland over the last 17,000 years, at 100 years intervals.
33
Fig. VII Examples of the main steps in the recovery of archaeobotanical remains.
36
Fig. 1.1 Geographical distribution of provenance regions of Q. ilex and P. halepensis included in
Chapter 1.
52
Fig. 1.2 Mean wood ∆13C in Q. ilex and P. halepensis trees grown in two slope aspects
(North/South) within each provenance region.
55
Fig. 1.3 Relationship between wood ∆13C in Q. ilex and P. halepensis and either mean annual
precipitation or the ratio between precipitation and potential evapotranspiration.
56
Fig. 1.4 Relationship between mean radial growth in Q. ilex and P. halepensis and either mean
annual precipitation or wood ∆13C.
57
Fig. 1.5 Correlation coefficients between wood ∆ C and radial growth and monthly values of
precipitation and potential evapotranspiration.
57
Fig. 2.1 Geographical distribution of the reference sites used to test the accuracy of climatic models
and the sampling sites for Pinus halepensis wood cores.
67
Fig. 2.2 Component weights plot of the principal components analysis including climatic variables
and δ13C and δ18O in different wood fractions.
72
Fig. 2.3 Correlation coefficients between seasonal means of climatic variables and δ13C and δ18O of
the different wood fractions.
73
Fig. 3.1 Geographical distribution of sampling locations for reference wood cores and
archaeological sites.
83
13
171
List of tables and figures
Figure description
Page
Fig. 3.2 Mean values and standard error for the δ13C and %C in intact wood and after carbonisation
at a range of temperatures.
86
Fig. 3.3 Relationship between ∆ C in wood and either annual precipitation or the ratio between
precipitation and evapotranspiration.
88
Fig. 3.4 Estimated evolution of annual precipitation and the ratio between precipitation and
evapotranspiration, according to the estimated ∆13C of original wood.
89
Fig. 4.1 Relationship between water inputs during grain filling and ∆13C of barley and wheat
grains.
104
Fig. 4.2 Estimation of past water inputs from ∆13C of archaeological cereal grains collected from
three areas of the Mediterranean Basin.
105
Fig. 4.3 Evolution of water inputs in archaeological cereal grains from NE Spain, compared with
average present values and with ∆13C of wood charcoal from SE France.
106
Fig. 4.4 Relationship between ∆13C values of different crops: detection of outliers for the
relationship between wheat and barley and cereal/legumes relationship.
107
Fig. 5.1 Regression models across treatments to estimate initial grain weight of wheat and barley
from the products Length x Breadth and Length x Thickness.
120
Fig. 5.2 Evolution of grain weight of barley and wheat in the Segre and Cinca Valley from Bronze
Age to Second Iron Age.
122
Fig. 6.1 Geographical location of the archaeological sites included in Chapter 6.
131
13
Fig. 6.2 Evolution of ∆13C and grain weight in wheat and barley during the last four millennia, and
residuals resulting from the linear regression between the two variables.
135
Fig. 6.3 Comparison between the evolution of estimated annual precipitation and total water inputs
during grain filling in cereals (wheat and barley combined).
137
Fig. VIII Relationship between climate and potential distribution of Q. ilex and P. halepensis in
Eastern Spain.
145
Fig. IX Comparison of the r2 between ∆13C in wood and monthly precipitation for Quercus ilex and
Pinus halepensis and proposed conceptual model.
147
Fig. X Relationship between ∆13C and δ18O in wheat grains and either total water inputs during
grain filling and average temperature throughout the growing season.
172

153